Affinity selected signature peptides for protein identification and quantification

ABSTRACT

A method for protein identification in complex mixtures that utilizes affinity selection of constituent proteolytic peptide fragments unique to a protein analyte. These “signature peptides” function as analytical surrogates. Mass spectrometric analysis of the proteolyzed mixture permits identification of a protein in a complex sample without purifying the protein or obtaining its composite peptide signature.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/203,227, filed May 5, 2000, U.S. Provisional Application Ser. No.60/208,372, filed May 31, 2000, and U.S. Provisional Application Ser.No. 60/208,184, filed May 31, 2000, each of which is incorporated hereinby reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under a grant from theNational Institutes of Health, Grant Nos. 25431 and GM 59996. The U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

DNA sequencing of the human genome has profoundly advanced ourunderstanding of the molecular anatomy of mammalian cells. However,knowing the sequence of all the genes in a cell and extrapolating fromthis the probable products a cell is capable of producing is not enough.It is clear that i) not all genes are expressed to the same degree; ii)the DNA sequence does not always tell you the structure of a protein inthe cases of post-transcriptional and post-translational modifications;iii) knowing the sequence of a gene tells you nothing about the controlof expression; iv) control of genetic expression is extremelycomplicated and can vary from protein to protein; v) post-translationalmodification can occur without de novo protein biosynthesis; and vi)variables other than genomic DNA can be responsible for disease.

In addition, it has recently become apparent that there is a poorcorrelation between genetic expression of mRNA, generally measured ascDNA, and the amount of protein expressed by that mRNA. Changes in mRNAconcentration are not necessarily proportional to changes in proteinconcentration. There are even many cases where mRNA will be up regulatedand protein concentration will not change at all. The steady stateconcentration of a protein can depend on the relative degree ofexpression from multiple genes and the activity of these gene productsin the synthesis of a specific protein. Glycoproteins provide a goodexample. The concentration of a glycoprotein can depend on the level towhich the gene coding for the polypeptide backbone is regulated, thepresence of all the enzymes responsible for the synthesis and attachmentof the oligosaccharide to the polypeptide, and the concentration ofglycosidases and proteases that degrade the glycoprotein. For thesereasons, analysis of regulation using messenger RNA-based techniquessuch as “DNA chips” alone is inadequate. It is clear that measuring theconcentration of mRNA that codes for the polypeptide backbone may eitherdistort or fail to recognize the total picture of how a protein isregulated. In cases where it is desirable to know how protein expressionlevels change, direct measurement of those levels may be needed.

Concentration and expression levels of specific proteins vary widely incells during the life cycle, both in absolute concentration and amountrelative to other proteins. Over- or under-expression are known to beindicators of genetic errors, faulty regulation, disease, or a responseto drugs. However, the small number of proteins that are up- ordown-regulated in response to a particular stimulus are difficult torecognize with current technology. Further, it is frequently difficultto predict which proteins are subject to regulation. The need to examine20,000 proteins in a cell to find the small number in regulator flux isa formidable problem. The ability to detect only the small numbers ofup- or down-regulated proteins in a complex protein milieu wouldsubstantially enhance the value of proteomics.

Proteins in complex mixtures are generally detected by some type offractionation or immunological assay technique. The advantages ofimmunological assay methods are their sensitivity, specificity forcertain structural features of antigens, low cost, and simplicity ofexecution. Immunological assays are generally restricted to thedetermination of single protein analytes. This means it is necessary toconduct multiple assays when it is necessary to determine small numbersof proteins in a sample. Hormone-receptor association, enzyme-inhibitorbinding, DNA-protein binding and lectin-glycoprotein association areother types of bioaffinity that have been exploited in proteinidentification, but are not as widely used as immunorecognition.Although not biospecific, immobilized metal affinity chromatography(IMAC) is yet another affinity method that recognizes a specificstructural element of polypeptides (J. Porath el al., Nature 258:598-599 (1975)).

The fractionation approach to protein identification in mixtures isoften more lengthy because analytes must be purified sufficiently toallow a detector to recognize specific features of the protein.Properties ranging from chemical reactivity to spectral characteristicsand molecular mass have been exploited for detection. Higher degrees ofpurification are required to eliminate interfering substances as thedetection mode becomes less specific. Since no single purification modecan resolve thousands of proteins, multidimensional fractionationprocedures must be used with complex mixtures. Ideally, the variousseparation modes constituting the multidimensional method should beorthogonal in selectivity. The two-dimensional (2D) gel electrophoresismethod of O'Farrell (J. Biol. Chem. 250:4007-4021 (1975)) is a goodexample. The first dimension exploits isoelectric focusing while thesecond is based on molecular size discrimination. At the limit, 6000 ormore proteins can be resolved. 2D gel electrophoresis is now widely usedin proteomics where it is the objective to identify thousands ofproteins in complex biological extracts.

The most definitive way to identify proteins in gels is by mass spectralanalysis of peptides obtained from a tryptic digest of the excised spot.Digestion of an excised spot with trypsin typically generates about30-200 peptides. Identification is greatly facilitated when peptidemolecular mass can be correlated with tryptic cleavage fragmentspredicted from a genomic database. Computer-assisted mathematicaldeconvolution algorithms are used to identify a protein based upon its“composite peptide signature.” Proteins can also be identified by theirseparation characteristics alone in some cases. The advantage of 2Delectrophoresis followed by tryptic mapping is that large numbers ofproteins can be identified simultaneously. However, the disadvantages ofthe technique are (1) it is very slow and requires a large number ofeither manual or robotic manipulations, (2) charged isoforms areresolved whereas uncharged variants in which no new charge is introducedare not, (3) proteins must be soluble to be examined, and (4)quantification by staining is poor.

In addition to being used to identify proteins, 2-D gel electrophoresishas also been used to assess relative changes in protein levels. Thedegree to which the concentration of a protein changes can be determinedby staining the gel and visually observing those spots that changed.Alternatively, changes in the concentration of a protein can bequantitated with a gel scanner. A control 2-D gel is required todetermine the concentration of the protein before it was either up ordown regulated. Tryptic cleavage of the excised spot and mass analysisusing mass spectrometry remains necessary to identify the protein whoseexpression level has changed.

Promising new techniques are emerging that replace 2-D gelelectrophoresis. Most involve some combination of high performanceliquid chromatography (HPLC) or capillary electrophoresis (CE) with massspectrometry to either create a “virtual 2-D gel” or go directly to thepeptide level of analysis by tryptic digesting all the proteins insamples as the initial step of analysis. The use of multidimensionalchromatography (MDC) to identify proteins in a complex mixture isfaster, easier to automate, and couples more readily to MS than 2D gelelectrophoresis. One of the more attractive features of chromatographicsystems is that they allow many dimensions of analysis to be coupled byanalyte transfer between dimensions through automated valve switching. Arecent report of an integrated six dimensional analytical system inwhich serum hemoglobin was purified and sequenced automatically in <2hours is an example (F. Hsieh et al., Anal. Chem. 68:455 (1996)).Subsequent to purification on an immunoaffinity column, hemoglobin wasdesorbed into an ion-exchange column for buffer exchange and thentryptic digested by passage through an immobilized trypsin column.Peptides eluting from the immobilized enzyme column were concentratedand desalted on a small, low-surface-area reversed-phase liquidchromatography (RPLC) column and then transferred to an analytical RPLCcolumn where they were separated and introduced into a mass spectrometerthrough an electrospray interface. Identification at the primarystructure level was achieved by a combination of chromatographicproperties and multidimensional mass spectrometry of the trypticpeptides. The ability of the immunosorbant to rapidly select the desiredanalyte for analysis was a great asset to this analysis. Size-exclusionor ion-exchange chromatography coupled to reversed-phase chromatographyare other examples of multidimensional systems, albeit of lowerselectivity than those using immunosorbant.

Although the methods described above are highly selective and widelyused, they have some attributes that limit their efficacy. One is theneed for proteins to be soluble before than can be analyzed. This can bea serious limitation in the case of membrane and structural proteinsthat are sparingly soluble. A second is that it is desirable or evennecessary in some cases for the protein analyte to be of nativestructure during at least part of the analysis. This is a limitationbecause it restricts the sample preparation protocol. Nativemacromolecular structures are notoriously more difficult to analyze thansmall molecules. The necessity for post separation protcolysis, as inthe 2D gel approach, is another limitation. Large numbers of fractionsmust be subjected to a 24 hour tryptic digestion protocol in theanalysis of a single sample when many proteins are being identified. Thetryptic digestion step is necessary because the mass of intact proteinsis far less useful in searching DNA databases than that of peptidesderived from the protein. And finally, pure proteins are a prerequisitefor antibody preparation in all the immunorecognition methods. Thepreparation of antibodies to an antigen is lengthy, laborious, andcostly, and many antigens have never been purified. This is particularlytrue of proteins predicted by genomic data alone. Purification iscomplicated by the fact that one does not know the degree to which aprotein is expressed, whether it is part of a multisubunit complex, orif it is post translationally modified.

Additionally, there is the issue of quantification. Measuring either therelative abundance of proteins or changes in protein concentrationremains a major challenge in proteomics. Improved methods for proteinidentification, quantification and detection of regulatory (or relativechange) or proteins, especially for the identification andquantification of proteins within a complex mixture, are clearly neededto advance the new science of proteomics.

SUMMARY OF THE INVENTION

The present invention provides a method for protein identification andquantification in complex mixtures that utilizes affinity selection ofconstituent peptide fragments. These peptides function as analyticalsurrogates for the proteins. The method of the invention makes itpossible to identify a protein in a sample, preferably a complex sample,without sequencing the entire protein. In many cases the method allowsfor identification of a protein in a sample without sequencing any partof the protein.

To “identify a protein” as that phrase is used herein means to determinethe identity of a protein or a class of proteins to which it belongs.Identifying a protein within a complex mixture of proteins involvesdetermining the presence or absence of a particular protein or class ofproteins in the mixture. Prior to identifying the protein according tothe method of the invention, it may be suspected that a particularprotein is in the mixture. On the other hand, the protein content of themixture may be largely unknown. Protein identification according to themethod may be used, for example, to catalog the contents of a complexmixture or to discover heretofore unknown proteins (e.g., proteins thatare predicted from the genome but have not yet been isolated).

Proteolysis of most proteins yields at least one unique “signaturepeptide.” The method of the invention identifies these constituentsignature peptides, preferably utilizing mass spectrometry, therebyallowing the protein comprising the signature peptide to bedistinguished from all other proteins in a complex mixture andidentified.

Constituent peptides can provide a generic signature for proteins aswell, especially when major portions of the amino acid sequence of aseries of protein variants are homologous. Glycoprotein variants thatdiffer in degree of glycosylation but not amino acid sequence are anexample. Proteins that have been modified by proteolysis are anothercase. Peptides that are unique to a variety of species of similarstructure are defined as “generic signature peptides”, and the inventionthus allows identification of a class of proteins by detecting andcharacterizing their generic signature peptides.

Proteins in a sample are initially fragmented, either as part of themethod or in advance of applying the method. Fragmentation in solutioncan be achieved using any desired method, such as by using chemical,enzymatic, or physical means. It should be understood that as usedherein, the terms “cleavage”, “proteolytic cleavage”, “proteolysis”,“fragmentation” and the like are used interchangeably and refer toscission of a chemical bond within peptides or proteins in solution toproduce peptide or protein “fragments” or “cleavage fragments.” Noparticular method of bond scission is intended or implied by the use ofthese terms. Fragmentation and the formation of peptide cleavagefragments in solution are to be differentiated from similar processes inthe gas phase within a mass spectrometer. These terms are contextspecific and relate to whether bond scission is occurring in solution orthe gas phase in a mass spectrometer.

Prior to proteolytic cleavage, the proteins are preferably alkylatedwith an alkylating agent in order to prevent the formation of dimers orother adducts through disulfide/dithiol exchange; optionally, theproteins are reduced prior to alkylation in order to facilitate thealkylation reaction and subsequent fragmentation. Some proteins areresistant to proteolysis unless they have been reduced and alkylatedprior to cleavage.

At least one peptide derived from the protein to be identifiedpreferably includes at least one affinity ligand. The affinity ligandcan be endogenous or exogenous. Preferably, the affinity ligand isendogeneous, thereby simplifying the method. If exogenous, the methodoptionally includes covalently attaching at least one affinity ligand toat least one protein (or peptide) in the sample before (or after)proteolytic cleavage. Optionally, the affinity ligand is covalentlylinked to the alkylating agent. The peptides are then contacted with acapture moiety to select peptides that contain the at least one affinityligand. If desired, a plurality of affinity ligands are attached, eachto at least one protein or peptide, and the peptides are contacted witha plurality of capture moieties to select peptides that contain at leastone affinity ligand. Optionally, the selected peptides are fractionatedat this point in order to further simplify the mixture and make itamenable to mass spectrometric analysis, yielding a plurality of peptidefractions.

Peptides are analyzed by mass spectrometry to detect at least onepeptide derived from the protein to be identified, thereby permittingidentification of the protein(s) from which the detected peptide wasderived. When the detected peptide is a signature peptide, the methodfurther includes determining the mass of the signature peptide and usingthe mass of the signature peptide to identify the protein from which thedetected peptide was derived. Optionally, the amino acid sequence of allor a portion of a detected peptide can be determined and used toidentify the protein from which the detected peptide was derived. In apreferred embodiment, the mass of the signature peptide is compared withthe masses of reference peptides derived from putative fragmentation ofa plurality of reference proteins in a database, wherein the masses ofthe reference peptides are adjusted to include the mass of the affinityligand, if necessary. Prior to making this comparison, referencepeptides are optionally computationally selected to exclude those thatdo not contain an amino acid upon which the affinity selection is basedin order to simplify the databases comparison.

The advantages of the method for protein identification of the inventionare numerous. Proteins themselves (which are large molecules compared topeptides) do not need to be separated electrophoretically orchromatographically, both time consuming steps. Moreover, affinityselection yields a subpopulation of peptides (typically eliminatingabout 90% of peptides) that is, advantageously, enriched for “signaturepeptides.” If desired, multiple selections can be used to produce theenriched, affinity-selected population, further simplifying the processof protein identification. In many cases, a protein can be identifiedfrom its signature peptides; it is not necessary to purify the protein,sequence any part of it, or determine its composite peptide signature inorder to identify it.

The present invention further provides a post-synthetic isotope labelingmethod useful for detecting differences in the concentration ofmetabolites between two samples. Application of the isotope labelingmethod of the invention is not limited to proteins, but can be used toidentify or quantitate other metabolites as well such as lipids, nucleicacids, polysaccharides, glycopeptides, glycoproteins, and the like. Thesamples are preferably complex mixtures, and the metabolite ispreferably a protein or a peptide. Advantageously, the method can beutilized with complex mixtures from various biological environments. Forexample, the method of the invention can be used to detect a protein orfamily of proteins that are in regulatory flux in response to theapplication of a stimulus. Peptides derived from these proteins exhibitsubstantially the same isotope ratios, which differ from the normalizedisotope ratio determined for proteins that are not in flux, indicatingthat they are co-regulated. Or, samples can be obtained from differentorganisms, cells, organs, tissues or bodily fluids, in which case themethod permits determination of the differences in concentration of atleast one protein in the organisms, cells, organs, tissues or bodilyfluids from which the samples were obtained.

The post-synthetic isotope labeling method of the invention involvesattaching a first chemical moiety to a protein, peptide, or the cleavageproducts of a protein in a first sample and a second chemical moiety toa protein, peptide, or the cleavage products of a protein in a secondsample to yield first and second isotopically labeled proteins, peptidesor protein cleavage products, respectively, that are chemicallyequivalent yet isotopically distinct. The chemical moiety can be asingle atom (e.g., oxygen) or a group of atoms (e.g., an acetyl group).The labeled proteins, peptides or peptide cleavage products areisotopically distinct because they contain different isotopic variantsof the same chemical entity (e.g, a peptide in the first sample contains¹H where the peptide in the second sample contains ²H; or a peptide inthe first sample contains ¹²C where the peptide in the second samplecontains ¹³C).

When a complex protein mixture is being analyzed, isotopic labeling canbe performed either before or after cleavage of the proteins.Preferably, isotopic labeling is performed after cleavage, and the firstand second chemical moieties are attached to at least one amino group,preferably the N-terminus, and/or at least one carboxylic acid group,preferably the C-terminus, on the peptides. Conveniently, the N-terminiof proteins or peptides can be labeled in an acetylation reaction,and/or the C-termini of proteins or peptides can be labeled byincorporation of ¹⁸O from H₂ ¹⁸O in the hydrolysis reaction. In thelatter case, one chemical moiety is represented by ¹⁶O, the naturallyoccurring isotope, and the other chemical moiety is represented by ¹⁸O;in effect, this particular process can be considered as “isotopicallylabeling” only one of the samples (the one that carries the ¹⁸Oisotope). When both the N-termini and the C-termini of proteins orpeptides are isotopically labeled, it is possible to differentiatebetween C-terminal peptides, N-terminal blocked peptides, and those thatare internal. Labeling both the N- and C-terminus of the proteins orpeptides also facilitates the analysis of single amino acidpolymorphisms. Labeling at the N- and/or C-terminus allows all orsubstantially all proteolytic peptides to be labeled, the advantages ofwhich are discussed below.

At least a portion of each sample is typically mixed together to yield acombined sample, which is subjected to mass spectrometric analysis.Control and experimental samples are mixed after labeling, fractionscontaining the desired components are selected from the mixture, andconcentration ratio is determined to identify analytes that have changedin concentration between the two samples. However, actual mixing of thesamples is not required, and the mass spectrometric analysis can becarried out on each sample independently, then analyzed with theassistance of a computer to achieve the same end. This important featureof the method significantly reduces processing time and facilitatesautomation of the process.

The members of at least one pair of chemically equivalent, isotopicallydistinct peptides optionally include at least one affinity ligand. Theaffinity ligand can be endogenous or exogenous. If exogenous, the methodoptionally includes covalently attaching at least one affinity ligand toat least one protein (or peptide) in the sample before (or after)proteolytic cleavage. Optionally, the affinity ligand is covalentlylinked to the alkylating agent. Prior to determining the isotope ratios,the peptides are contacted with a capture moiety to select peptideswhich contain the at least one affinity ligand. If desired, a pluralityof affinity ligands can be attached, each to at least one protein orpeptide, and the peptides are contacted with a plurality of capturemoieties to select peptides that contain at least one affinity ligand.In a preferred embodiment, at least one “signature peptide” unique to aprotein is selected, and the signature peptide is subsequently used toidentify the protein from which it was derived.

In a preferred embodiment, the affinity ligand is distinct from theisotope labeling moieties. In other words, the labeling step is notcoupled to the selection step. This allows the quantitation function andthe selection function to be independent of one another, permitting morefreedom in the choice of reagents and labeling sites and also allowingan isotopically labeled sample to be assayed for different signaturepeptides. Another advantage of uncoupling the labeling and selectionsteps is that labeling, if performed after cleavage, can be applied in amanner to label all peptides, not just the peptide to be selected.

When the method involves labeling all peptide fragments, it is referredto herein as the global internal standard technology (GIST) method (FIG.1). Components from control samples function as standards against whichthe concentration of components in experimental samples are compared.When the differential labeling process is directed at primary amine,carboxyl groups, or both in peptides produced during proteolysis of theproteome, an internal standard is created for essentially every peptidein the mixture. Possible, but rare, exceptions to this include peptidesthat are derivatized or blocked on the N-terminus or C-termninus.Examples of N-terminal blocking include f-met proteins found inbacterial systems, acylation of serum proteins, and the formation of thecyclic moiety pyrrolidone carboxylic acid (pyroGlu or pGlu) at anN-terminal glutamate. The C-terminus can be blocked due to the formationof an amide or an ester; for example many prenylated proteins areblocked at the C-terminus with a methyl ester. In any event, becausevirtually all peptide fragments in the sample are labeled, the method isreferred to as a global labeling strategy. This global internal standardtechnology (GIST) for labeling may be used to quantifying the relativeconcentration of all components in it complex mixtures.

As an example, an investigator can isotopically label all peptides (bylabeling the free amino group or the free carboxyl group thatcharacterizes nearly every peptide), then independently affinity labelthe isotopically labeled peptides at other sites, either in parallel orin series. Perhaps tyrosines in an aliquot of a globally isotopicallylabeled peptide pool could be affinity labeled (either before or afterprotein fragmentation), after which peptides containing tyrosines couldbe selected. Then, another aliquot of the same peptide pool could beselected for histidine-containing peptides. Alternatively, the selectedtyrosine-containing peptide subpopulation could be further selected forhistidine, depending on the interests of the investigator. Isotoperatios for any of these selected peptides could be determined using massspectrometry. See Example V for examples of multiple selections onglobally isotopically labeled peptides.

Although the advantages of keeping the isotopic labeling stepindependent of the selection criteria are significant and very clear, itshould nonetheless be understood that, if desired, the affinity ligandand the first and second moieties used to isotopically label thepeptides or proteins can be the same, as in the case where proteins orpeptide are affinity labeled at cysteine with isotopically distinctforms of the alkylating agent, iodoacetic acid, coupled to the affinityligand biotin. It is significant that if cysteine-containing peptidesare to be selected, the investigator is generally limited toderivatizing the protein prior to cleavage, as part of the reduction andalkylation process. In addition, it should be cautioned that wheneverisotopically labeling is coupled to the selection process, only asubpopulation of the peptide fragments will be isotopically labeled.Moreover, only one selection criterion can be effectively used forcomparative quantitative analysis of peptides. Application of a secondselection criterion selects for peptides that are not necessarilyisotopically labeled, rendering quantitative comparison impossible. If asecond selection is desired, the protein or peptide sample must beisotopically labeled a second time with the new derivatizing agent.

Furthermore, unless peptides are globally labeled isotopically, it isnot possible to select and quantitatively compare peptides on the basisof an inherent feature of the peptide (i.e., an endogenous affinityligand). For example, tyrosinephosphate-containing peptides selectedusing immunochromatrography, or histidine-containing peptides selectedusing IMAC (see below) could not be quantitatively compared unless aglobal isotopic labeling strategy was used. Selection using anendogenous affinity ligand (as opposed to an exogenous ligand that needsto be linked to the peptide in a separate step) is preferred in themethod of the invention, therefore the ability to globally label thepeptides is an extremely important and useful aspect of the invention.

Optionally in the method of the invention, at some point prior todetermining the isotope ratios, the combined peptide sample isfractionated, for example using a chromatographic or electrophoretictechnique, to reduce its complexity so that it is amenable to massspectrometric analysis, yielding at least one fraction containing theisotopically labeled first and second proteins and/or peptides.

During mass spectrometric analysis, a normalized isotope ratiocharacterizing metabolites whose concentration is the same in the firstand second samples is first determined, then the isotope ratio of thefirst and second isotopically labeled metabolites is determined andcompared to the normalized isotope ratio. A difference in the isotoperatio of the first and second isotopically labeled metabolites and thenormalized isotope ratio is indicative of a difference in concentrationof the metabolite in the first and second samples.

When the metabolites are affinity-labeled peptides derived from aprotein, mass spectrometric analysis can be used to detect at least onepeptide and identify the protein from which the detected peptide wasderived. When the detected peptide is a signature peptide, the methodpreferably includes determining the mass of the signature peptide andusing the mass of the signature peptide to identify the protein fromwhich the detected peptide was derived. The invention thus makes itpossible to identify a protein in a sample, preferably a complex sample,without sequencing the entire protein. In many cases the method allowsfor identification of a protein in a sample without sequencing any partof the protein. In a preferred embodiment, the mass of the signaturepeptide compared with the masses of reference peptides derived fromputative proteolytic cleavage of a plurality of reference proteins in adatabase, wherein the mass of the references peptides are adjusted toinclude the mass of the affinity ligand, if necessary. Prior to makingthis comparison, reference peptides are optionally computationallyselected to exclude those that do not contain an amino acid upon whichthe affinity selection is based in order to simplify the databasecomparison. Optionally, the amino acid sequence of the detected peptidecan be determined and used to identify the protein from which thedetected peptide was derived.

When a protein or peptide is present in a one sample but not in anothersample, it can be difficult to determine which sample generated thesingle peak observed during mass spectrometric analysis of the combinedsample. This problem is addressed by double labeling the first sample,either before or after proteolytic cleavage, with two different isotopesor two different numbers of heavy atoms. The first sample is partitionedinto first and second subsamples, which are labeled with chemicallyequivalent moieties containing first and second isotopes or numbers ofheavy atoms, respectively. Polypeptides in the second sample are labeledwith a chemically equivalent moiety containing a third isotope or numberof heavy atoms greater than in the other two cases. The first, secondand third labeling agents are chemically equivalent yet isotopicallydistinct. Preferably, the labeling agents are acylating agents. Thethree samples are combined and optionally fractionating to yield aplurality of peptide fractions amenable to mass spectrometric isotoperatio analysis. The presence of a doublet during mass spectrometricanalysis due to the presence of the first and second isotope labelingagents indicates the absence of the protein in the second sample, andthe presence of a single peak due to the presence of the third isotopelabeling agent indicates the absence of the protein in the first sample.

Sometimes a solution based fragmentation of a protein mixture generatestwo or more different peptides having identical mass and chromatographicseparation properties (“isobaric peptides”), such as peptides with thesame amino acid composition but different amino acid sequences. In thiscase, the composite mass spectrum will not reflect the isotope ratios ofthe individual peptides. However, the mass of one or more of theconstituent fragment ions generated during gas phase fragmentation ofthe peptide will be different. These fragment ions can therefore beresolved by subjecting the precursor ions to a second dimension of massspectrometry, provided the peptides are isotopically labeled at eitherthe N - or the C-terminus. Isotopic peaks from the first dimensionspanning a mass range of up to about 20 amu are selected for massspectrometric analysis in the second dimension. Fragmentation prior tothe second dimension of mass spectrometry can occur by eitherpost-source decay or collision-induced (or collision-activated)dissociation (CID or CAD) of the precursor ion. The isotope ratio ofthose fragment ions that differ between peptides can be used to quantifythe peptides.

This problem is not limited to isobaric peptides. When the differencebetween the masses of the labeling agents is 3 amu a problem will occurany time the peptide clusters are within 6 amu of each other such thatthey overlap. A range of isotope peaks, for example about 6 to about 10amu range for deuterium labeled peptides, is selected for massspectrometric analysis in the second dimension, and unique fragment ionscan be located. When a broader mass window is selected for use in thesecond dimension for deuterated samples, ²H₃ and ¹H₃ N-acetyl labeledforms of the peptide will both be present in the second dimension, andthe ²H₃ and ¹H₃ labeling will only be found on the fragment ions thatcontain portions of the molecule that were acetylated. Quantificationcan be achieved by measuring the ²H₃ and ¹H₃ ratio in the seconddimension.

The methods for protein identification and, optionally, quantificationdescribed herein offer the investigator a high degree of experimentalflexibility and are also very amenable to automation. They are, inaddition, extremely sensitive; for example, the use of mass spectrometryto uniquely define the signature peptide (by its mass) makes it possiblefor the isotope labeling method of the invention to distinguish amongsingle site protein polymorphisms.

It should be noted that, while isotope labeling of the proteins orconstituent peptides is useful for quantification and quantitativecomparison of proteins and/or peptides in a complex mixture, isotopelabeling is not necessary to identify proteins in a complex mixture. Aprotein can be identified by comparing the mass of a signature peptideto the masses of peptides in a peptide database formed fromcomputational cleavage of a set of proteins. The absence of the need toisotopically label the protein or peptides facilitates automation andalso makes protein identification using database searching algorithmseasier, since the peptides do not include the mass of an exogenousisotope labeling reagent.

The terms “a”, “an”, “the”, and “at least one” include the singular aswell as the plural unless specified to the contrary.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of coupled and uncoupled methods ofthe invention.

FIG. 2 is a reversed-phase chromatogram of proteins isolated from bovinenuclei by chromatography on a Bandeiraea simplicifolia (BS-II) lectinaffinity column. Elution was achieved using a 0.20 M solution ofN-acetylglucosamine.

FIG. 3 is a reversed-phase chromatogram of tryptic digestedglycopeptides isolated from bovine nuclei by chromatography on a BS-IIlectin affinity column. Elution was achieved using a 0.20 M solution ofN-acetylglucosamine.

FIG. 4(a)-(d) shows mass spectra of various glycopeptide fractionscollected from the reversed phase column.

FIG. 5 is a reversed-phase chromatogram of (a) a peptide map of humanserotransferrin and (b) two human serotransferrin glycopeptides isolatedfrom a conconavalin A column.

FIG. 6 is a matrix-assisted laser desorption ionization-time of flight(MALDI-TOF mass spectrum of (a) the first glycopeptide from humanserotransferrin and (b) the second glycopeptide from humanserotransferrin.

FIG. 7 is a reversed-phase chromatogram of (a) glycopeptides isolatedfrom human serum and (b) glycopeptides isolated from human serum.

FIG. 8 is a mass spectrum of fractions isolated from human serumcontaining (a) the first glycopeptide from human serotransferrin and (b)the second glycopeptide from human serotransferrin.

FIG. 9 is a MALDI-mass spectrum of a deuterium labeled peptidecontaining four lysines.

FIG. 10 is a MALDI-TOF mass spectrum of (a) labeled and unlabeledlysine-containing peptide in negative mode detection and (b) alysine-containing peptide detected in positive mode.

FIG. 11 is a MALDI mass spectrum of a peptide that contains (a) lysineand (b) arginine.

DETAILED DESCRIPTION OF THE INVENTION

Roughly 90% of the time, the amino acid sequence of a peptide fragmenthaving a mass of over 500 daltons will be unique to the protein fromwhich it is derived. This varies somewhat with the organism. Because ofthis uniqueness, these peptides are referred to herein as “signaturepeptides.” Signature peptides are often, but not always, characterizedby features such as low abundance amino acids such as cysteine orhistidine, phosphorylation or glycosylation, and antigenic properties.If one were to select from a pool of all tryptic peptides produced fromproteolysis of the proteome those peptides that contain the lowabundance amino acids histidine or cysteine, there would be between oneand four “signature peptides” per protein. The number depends to someextent on the size of the protein.

A signature peptide is a peptide that is unique to a single protein andpreferably contains about 6 to about 20 amino acids. Enzymatic digestionof a complex mixture of proteins will therefore generate peptides,including signature peptides, that can theoretically be used to identifyparticular proteins in the complex mixture. Indeed, liquidchromatography, capillary electrophoresis, and mass spectrometry aremuch more adept at the analysis of peptides than the intact proteinsfrom which they are derived. A complex mixture of proteins preferablycontains at least about 100 proteins, more preferably it contains atleast about 1000 proteins and it can contain several thousand proteins.However, when a complex mixture containing thousands of proteins isproteolytically digested, it is probable that a hundred thousand or morepeptides will be generated during proteolysis. This is beyond theresolving power of liquid chromatography and mass spectrometry systems.

This problem is solved in the present invention by utilizing aselection, preferably an affinity selection, after the proteolyticcleavage to select peptide fragments that contain specific amino acids,thereby substantially reducing the number of sample components that mustbe subjected to further analysis. The method for protein identificationof the invention is well-suited to the identification of proteins in acomplex mixture, and at a minimum includes proteolytic cleavage of aprotein and affinity selection of the peptides. The affinity selectioncan be effected using an affinity ligand that has been covalentlyattached to the protein (prior to cleavage) or its constituent peptides(after cleavage), or using an endogenous affinity ligand. The affinityselection is preferably based on low abundance amino acids orpost-translational modifications so as to preferentially isolate“signature peptides.” The method is not limited by the affinityselection method(s) employed and nonlimiting examples of affinityselections are described herein and can also be found in the scientificliterature, for example in M. Wilchek, Meth. Enzymol. 34, 182-195(1974). This approach enormously reduces the complexity of the mixture.If desired, two or more affinity ligands (e.g., primary and secondaryaffinity ligands) can be used, thereby allowing a finer selection.Illustrative examples of pre- and post-digestion labeling are shown inExamples IV and V, below.

Preferably, the affinity selected peptides are subjected to afractionation step to reduce sample size prior to the determination ofpeptide masses. A premise of the signature peptide strategy is that manymore peptides are generated during proteolysis than are needed forprotein identification. This assumption means that large numbers ofpeptides potentially can be eliminated, while still leaving enough forprotein identification.

The method is not limited by the techniques used for selection and/orfractionation. Typically, fractionation is carried out using single ormultidimensional chromatography such as reversed phase chromatography(RPC), ion exchange chromatography, hydrophobic interactionchromatography, size exclusion chromatography, or affinity fractionationsuch as immunoaffinity and immobilized metal affinity chromatography.Preferably the fractionation involves surface-mediated selectionstrategies. Electrophoresis, either slab gel or capillaryelectrophoresis, can also be used to fractionate the peptides. Examplesof slab gel electrophoretic methods include sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and native gelelectrophoresis. Capillary electrophoresis methods that can be used forfractionation include capillary gel electrophoresis (CGE), capillaryzone electrophoresis (CZE) and capillary electrochromatography (CEC),capillary isoelectric focusing, immobilized metal affinitychromatography and affinity electrophoresis.

Masses of the affinity-selected peptides, which include the “signaturepeptides,” are preferably determined by mass spectrometry, preferablyusing matrix assisted laser desorption ionization (MALDI) orelectrospray ionization (ESI), and mass of the peptides is analyzedusing time-of-flight (TOF), quadrapole, ion trap, magnetic sector or ioncyclotron resonance mass analyzers, or a combination thereof including,without limitation, TOF-TOF and other combinations. Preferably the massof the peptides is determined with a mass accuracy of about 10 ppm orbetter; more preferably, masses are determined with a mass accuracy ofabout 5 ppm or better; most preferably they are determined with a massaccuracy of about 1 ppm or better. The lower the ppm value, the moreaccurate the mass determination and the less sequence data is needed forpeptide identification.

It should be understood that the term “protein,” as used herein, refersto a polymer of amino acids and does not connote a specific length of apolymer of amino acids. Thus, for example, the terms oligopeptide,polypeptide, and enzyme are included within the definition of protein,whether produced using recombinant techniques, chemical or enzymaticsynthesis, or naturally occurring. This term also includes polypeptidesthat have been modified or derivatized, such as by glycosylation,acetylation, phosphorylation, and the like. When the term “peptide” isused herein, it generally refers to a protein fragment produced insolution.

Selection of Sample

The method of the invention is designed for use in complex samplescontaining a number of different proteins. Preferably the samplecontains at least about two proteins; more preferably it contains atleast about 100 proteins; still more preferably it contains at leastabout 1000 proteins. A sample can therefore include total cellularprotein or some fraction thereof. For example, a sample can be obtainedfrom a particular cellular compartment or organelle, using methods suchas centrifugal fractionation. The sample can be derived from any type ofcell, organism, tissue, organ, or bodily fluid, without limitation. Themethod of the invention can be used to identify one or more proteins inthe sample, and is typically used to identify multiple proteins in asingle complex mixture. It should therefore be understood that when themethod of the invention is referred to, for simplicity, as a method foridentifying “a protein” in a mixture that contains multiple proteins,the term “a protein” is intended to mean “at least one protein” and thusincludes one or more proteins.

Fragmentation of Proteins

Fragmentation of proteins can be achieved by chemical, enzymatic orphysical means, including, for example, sonication or shearing.Preferably, a protease enzyme is used, such as trypsin, chymotrypsin,papain, gluc-C, endo lys-C, proteinase K, carboxypeptidase, calpain,subtilisin and pepsin; more preferably, a trypsin digest is performed.Alternatively, chemical agents such as cyanogen bromide can be used toeffect proteolysis. The proteolytic agent can be immobilized in or on asupport, or can be free in solution.

Selecting Peptides with Specific Amino Acids

Peptides from complex proteolytic digests that contain low abundanceamino acids or specific post-translational modifications are selected(purified) to reduce sample complexity while at the same time aiding inthe identification of peptides selected from the mixture. Selection ofpeptide fragments that contain cysteine, tryptophan, histidine,methionine, tyrosine, tyrosine phosphate, serine and threoninephosphate, O-linked oligosaccharides, or N-linked oligosaccharides, orany combination thereof can be achieved. It is also possible todetermine whether the peptide has a C-terminal lysine or arginine and atleast one other amino acid.

The present invention thus provides for selection of proteolyticcleavage fragments that contain these specific amino acids orpost-translational modifications, and includes a method of purifyingindividual peptides sufficiently that they are amenable to MALDI massspectrometry (MALDI-MS). In view of the fact that MALDI-MS canaccommodate samples with 50-150 peptides and a good reversed phasechromatography (RPC) column can produce 200 peaks, a high qualityRPC-MALDI-MS system can be expected to analyze a mixture of 10,000 to30,000 peptides. Preliminary studies by others with less powerfulRPC-electrospray-MS systems support this conclusion (F. Hsieh et al.,Anal. Chem. 70:1847-1852 (1998)). Selection of ten or less peptides fromeach protein would allow this system to deal with mixtures of 1,000 to3,000 proteins in the worst case scenario. More stringent selectionwould increase this number. The selection method chosen is thus veryimportant.

Affinity Tags

An affinity tag used for selection can be endogenous to the protein, orit can be added by chemical or enzymatic processes. The term “affinitytag,” as used herein, refers to a chemical moiety that functions as, orcontains, an affinity ligand that is capable of binding (preferablynoncovalently, but covalent linkages are contemplated also) to a second,“capture” chemical moiety, such that a protein or peptide that naturallycontains or is derivatized to include the affinity tag can be selected(or “captured”) from a pool of proteins or peptides by contacting thepool with the capture moiety. The capture moiety is preferably bound toa support surface, preferably a porous support surface, as a stationaryphase. Examples of suitable supports include porous silica, poroustitania, porous zirconia, porous organic polymers, porouspolysaccharides, or any of these supports in non-porous form.

Preferably the interactions between the affinity tag and the capturemoiety are specific and reversible (e.g., noncovalent binding orhydrolyzable covalent linkage), but they can, if desired, initially be,or subsequently be made, irreversible (e.g., a nonhydrolyzable covalentlinkage between the affinity tag and the capture moiety). It isimportant to understand that the invention is not limited to the use ofany particular affinity ligand.

Examples of endogenous affinity ligands include naturally occurringamino acids such as cysteine (selected with, for example, an acylatingreagent) and histidine, as well as carbohydrate and phosphate moieties.A portion of the protein or peptide amino acid sequence that defines anantigen can also serve as an endogenous affinity ligand, which isparticularly useful if the endogenous amino acid sequence is common tomore than one protein in the original mixture. In that case, apolyclonal or monoclonal antibody that selects for families ofpolypeptides that contain the endogenous antigenic sequence can be usedas the capture moiety. An antigen is a substance that reacts withproducts of an immune response stimulated by a specific immunogen,including antibodies and/or T lymphocytes. As is known in the art, anantibody molecule or a T lymphocyte may bind to various substances, forexample, sugars, lipids, intermediary metabolites, autocoids, hormones,complex carbohydrates, phospholipids, nucleic acids, and proteins. Asused herein, the term “antigen” means any substance present in a peptidethat may be captured by binding to an antibody, a T lymphocyte, thebinding portion of an antibody or the binding portion of T lymphocyte.

A non-endogenous (i.e., exogenous) affinity tag can be added to aprotein or peptide by, for example, first covalently linking theaffinity ligand to a derivatizing agent to form an affinity tag, thenusing the affinity tag to derivatize at least one functional group onthe protein or peptide. Alternatively, the protein or peptide can befirst derivatized with the derivatizing agent, then the affinity ligandcan be covalently linked to the derivatized protein or peptide at a siteon the derivatizing agent. An example of an affinity ligand that can becovalently linked to a protein or peptide by way chemical or enzymaticderivatization is a peptide, preferably a peptide antigen orpolyhistidine. A peptide antigen can itself be derivatized with, forexample, a 2,4-dinitrophenyl or fluorescein moiety, which renders thepeptide more antigenic. A peptide antigen can be conveniently capturedby an immunosorbant that contains a bound monoclonal or polyclonalantibody specific for the peptide antigen. A polyhistidine tag, on theother hand, is typically captured by an IMAC column containing a metalchelating agent loaded with nickel or copper. Biotin, preferablyethylenediamine terminated biotin, which can be captured by the naturalreceptor avidin, represents another affinity ligand. Other naturalreceptors can also be used as capture moieties in embodiments whereintheir ligands serve as affinity ligands. Other affinity ligands includedinitrophenol (which is typically captured using an antibody or amolecularly imprinted polymer), short oligonucleotides, and polypeptidenucleic acids (PNA) (which are typically captured by nucleic acidhybridization). Molecularly imprinted polymers can also be used tocapture. The affinity ligand is typically linked to a chemical moietythat is capable of derivatizing a selected functional group on a peptideor protein, to form an affinity tag. An affinity ligand can, forexample, be covalently linked to maleimide (a protein or peptidederivatizing agent) to yield an affinity tag, which is then used toderivative the free sulfhydryl groups in cysteine, as further describedbelow.

Selecting Cysteine-containing Peptides

It is a common strategy to alkylate the sulfhydryl groups in a proteinbefore proteolysis. Alkylation is generally based on two kinds ofreactions. One is to alkylate with a reagent such as iodoacetic acid(IAA) or iodoacetamide (IAM). The other is to react with vinyl pyridine,maleic acid, or N-ethylmaleimide (NEM). This second derivatizationmethod is based on the propensity of —SH groups to add to the C═C doublebond in a conjugated system. Alkylating agents linked to an affinityligand double as affinity tags and can be used to select cysteinecontaining peptides after, or concomitant with, alkylation. For example,affinity-tagged iodoacetic acid is a convenient selection for cysteine.

Optionally, the protein is reduced prior to alkylation to convert allthe disulfides (cystines) into sulfhydryls (cysteines) prior toderivatization. Alkylation can be performed either prior to reduction(permitting the capture of only those fragments in which the cysteine isfree in the native protein) or after reduction (permitting capture ofthe larger group containing all cysteine-containing peptides, includethose that are in the oxidized cystine form in the native protein).

Preparation of an affinity tagged N-ethylmaleimide may be achieved bythe addition of a primary amine-containing affinity tag to maleicanhydride. The actual affinity tag may be chosen from among a number ofspecies ranging from peptide antigens, polyhistidine, biotin,dinitrophenol, or polypeptide nucleic acids (PNA). Peptide anddinitrophenol tags are typically selected with an antibody whereas thebiotin tag is selected with avidin. When the affinity tag includes asthe affinity ligand a peptide, and when proteolysis of the proteinmixture is accomplished after derivatization using trypsin or lys-C, thepeptide affinity ligand preferably does not contain lysine or arginine,so as to prevent the affinity ligand from also being cleaved duringproteolysis. Biotin is a preferred affinity ligand because it isselected with very high affinity and can be captured with readilyavailable avidin/streptavidin columns or magnetic beads. As noted above,polyhistidine tags are selected in an immobilized metal affinitychromatography (IMAC) capture step. This selection route has theadvantage that the columns are much less expensive, they are of highcapacity, and analytes are easily desorbed.

Alternatively, cysteine-containing peptides or proteins can be captureddirectly during alkylation without incorporating an affinity ligand intothe alkylating agent. An alkylating agent is immobilized on a suitablesubstrate, and the protein or peptide mixture is contacted with theimmobilized alkylating agent to select cysteine-containing peptides orproteins. If proteins are selected, proteolysis can be convenientlycarried out on the immobilized proteins to yield immobilizedcysteine-containing peptides. Selected peptides or proteins are thenreleased from the substrate and subjected to further processing inaccordance with the method of the invention.

When alkylation is done in solution, excess affinity tagged alkylatingagent is removed prior to selection with an immobilized capture moiety.Failure to do so will severely reduce the capacity of the capturesorbent. This is because the tagged alkylating agent is used in greatexcess and the affinity sorbent cannot discriminate between excessreagent and tagged peptides. This problem is readily circumvented byusing a small size exclusion column to separate alkylated proteins fromexcess reagent prior to affinity selection. The whole process can beautomated (as further described below) by using a multidimensionalchromatography system with, for example, a size exclusion column, animmobilized trypsin column, an affinity selector column, and a reversedphase column. After size discrimination the protein is valved throughthe trypsin column and the peptides in the effluent passed directly tothe affinity column for selection. After capture and concentration onthe affinity column, tagged peptides are desorbed from the affinitycolumn and transferred to the reversed phase column where they wereagain captured and concentrated. Finally, the peptides are eluted with avolatile mobile phase and fractions collected for mass spectralanalysis. Automation in this manner has been found to work well.

Selecting Tyrosine-containing Peptides

Like cysteine, tyrosine is an amino acid that is present in proteins inlimited abundance. It is known that diazonium salts add to the aromaticring of tyrosine ortho to the hydroxyl groups; this fact has been widelyexploited in the immobilization of proteins through tyrosine.Accordingly, tyrosine-containing peptides or proteins can beaffinity-selected by derivatizing them with a diazonium salt that hasbeen coupled at its carboxyl group to a primary amine on an affinityligand, for example through the α-amino group on a peptide tag asdescribed above. Alternatively, that diazonium salt can be immobilizedon a suitable substrate, and the protein or peptide mixture is contactedwith the immobilized diazonium salt to select tyrosine-containingpeptides or proteins. If proteins are selected, proteolysis can beconveniently carried out on the immobilized proteins to yieldimmobilized tyrosine-containing peptides. Selected peptides or proteinsare then released from the substrate and subjected to further processingin accordance with the method of the invention.

Selecting Tryptophan-containing Peptides

Tryptophan is present in most mammalian proteins at a level of <3%. Thismeans that the average protein will yield only a few tryptophancontaining peptides. Selective derivatization of tryptophan has beenachieved with 2,4-dinitrophenylsulfenyl chloride at pH 5.0 (M. Wilchecket al., Biochem. Biophys. Acta 278:1-7 (1972)). Using an antibodydirected against 2,4-dinitrophenol, an immunosorbant was prepared toselect peptides with this label. The advantage of tryptophan selectionis that the number of peptides will generally be small.

Selecting Histidine-containing Peptides.

In view of the higher frequency of histidine in proteins, it would seemat first that far too many peptides would be selected to be useful. Thegreat strength of the procedure outlined below is that it selects on thebasis of the number of histidines, not just the presence of histidine.Immobilized metal affinity chromatography (IMAC) columns loaded withcopper easily produce ten or more peaks. The fact that a few other aminoacids are weakly selected is not a problem, and the specificity ofhistidine selection can, if desired, be greatly improved by acetylationof primary amino groups. Fractions from the IMAC column are transferredto an RPC-MALDI/MS system for analysis. The number of peptides that canpotentially be analyzed jumps to 100,000-300,000 in the IMAC approach.An automated IMAC-RPC-MALDI/MS system essentially identical to that usedfor cysteine selection has been assembled. The only difference is insubstituting an IMAC column for the affinity sorbent and changes in theelution protocol. Gradient elution in these systems is most easilyachieved by applying step gradients to the affinity column. Afterreduction, alkylation, and digestion, the peptide mixture is captured onthe IMAC column loaded with copper. Peptides are isocratically elutedfrom the IMAC using imidazole or a change in pH, and directlytransferred to the RPC column where they are concentrated at the head ofthe column. The IMAC is then taken of line, the solvent lines of theinstrument purged at 10 ml/minute for a few seconds with RPC solvent A,and then the RPC column is gradient eluted and column fractionscollected for MALDI-MS. When this is done, the RPC column is recycledwith the next solvent for step elution of the IMAC column, the IMACcolumn is then brought back on line, and the second set of peptides isisocratically eluted from the IMAC column and transferred to the RPCcolumn where they are readsorbed. The IMAC column is again takenoff-line, the system purged, and the second set of peptides is elutedfrom the RPC column. This process is repeated until the IMAC column hasbeen eluted. Again, everything leading up to MALDI-MS is automated.

Selecting Post-translationally Modified Proteins.

Post-translational modification plays an important role in regulation.For this reason, it is necessary to have methods that detect specificpost-translational modifications. Advantageously, the method of theinvention can distinguish among proteins having a single signaturepeptide where speciation occurs by post-translational modification, ifthe affinity ligand is associated with, or constitutes, thepost-translational moiety (e.g., sugar residue or phosphate). Among themore important post-translational modifications are i) thephosphorylation of tyrosine, serine, or threonine; ii) N-glycosylation;and iii) O-glycosylation.

Selecting Phosphoproteins

In the case of phosphorylated proteins, such as those containingphosphotyrosine and phosphoserine, selection can achieved withmonoclonal antibodies that target specific phosphorylated amino acids.For example, immunosorbant columns loaded with a tyrosine phosphatespecific monoclonal antibody are commercially available. Preferably, allproteins in a sample are digested, then the immunosorbant is used toselect only the tyrosine phosphate containing peptides. As in otherselection schemes, these peptides can separated by reversed phasechromatography and subjected to MALDI.

Alternatively, selection of phosphopeptides can be achieved using IMACcolumns loaded with gallium (M. Posewitz et al., Anal. Chem.71(14):2883-2992 (1999)). Phosphopeptides can also be selected usinganion exchange chromatography, preferably on a cationic support surface,at acidic pH.

In addition, because zirconate sorbents have high affinity for phosphatecontaining compounds (C. Dunlap et al., J. Chromatogr. A 746:199-210(1996)), zirconia-containing chromatography is expected to be suitablefor the purification of phosphoproteins and phosphopeptides. Zirconateclad silica sorbents can be prepared by applying zirconyl chloridedissolved in 2,4-pentadione to 500 angstrom pore diameter silica andthen heat treating the support at 400° C. Another alternative is theporous zirconate support recently described by Peter Carr (C. Dunlap etal., J. Chromatogr. A 746:199-210 (1996)). Phosphopeptides are elutedusing a phosphate buffer gradient. In many respects, this strategy isthe same as that of the IMAC columns.

Selecting O-linked Oligosaccharide Containing Peptides

Glycopeptides can be selected using lectins. For example, lectin fromBandeiraea simplicifolia (BS-II) binds readily to proteins containingN-acetylglucosamine. This lectin is immobilized on a silica support andused to affinity select O-glycosylated proteins, such transcriptionfactors, containing N-acetylglucosamine and the glycopeptides resultingfrom proteolysis. The protocol is essentially identical to the otheraffinity selection methods described above. Following reduction andalkylation, low molecular weight reagents are separated from proteins.The proteins are then tryptic digested, the glycopeptides selected onthe affinity column, and then the glycopeptides resolved by RPC. In thecase of some transcription factors, glycosylation is homogeneous andMALDI-MS of the intact glycopeptide is unambiguous. That is not the casewith the more complex O-linked glycopeptides obtained from many othersystems. Heterogeneity of glycosylation at a particular serine willproduce a complex mass spectrum that is difficult to interpret.Enzymatic deglycosylation of peptides subsequent to affinity selectionis indicated in these cases. Deglycosylation can also be achievedchemically with strong base and is followed by size exclusionchromatography to separate the peptides from the cleavedoligosaccharides.

It is important to note that O-linked and N-linked glycopeptides areeasily differentiated by selective cleavage of serine linkedoligosaccharides (E. Roquemore et al., Meth. Enzymol. 230:443-460(1994)). There are multiple ways to chemically differentiate betweenthese two classes of glycopeptides. For example, basic conditions inwhich the hemiacetal linkage to serine is readily cleaved can beutilized. In the process, serine is dehydrated to form an α,βunsaturated system (C═C—C═O). The C═C bond of this system may be eitherreduced with NaBH₄ or alkylated with a tagged thiol for further affinityselection. This would allow O-linked glycopeptides to be selected in thepresence of N-linked glycopeptides. The same result could be achievedwith enzymatic digestion.

Selecting N-linked Oligosaccharide-containing Peptides

As with O-linked oligosaccharide-containing peptides, lectins can beused to affinity select N-linked glycopeptides following reductivealkylation and proteolysis. To avoid selecting O-linked glycopeptides,the peptide mixture is subjected to conditions that cause selectivecleavage O-linked oligosaccharides prior to affinity selection using thelectin. Preferably O-linked deglycosylation is achieved using a basetreatment after reductive alkylation, followed by size exclusionchromatography to separate the peptides from the cleavedoligosaccharides. To address the potential problem of heterogeneity ofglycosylation, and N-linked glycopeptides are deglycosylated afterselection. Automation can be achieved with immobilized enzymes, but longresidence times in the enzyme columns are needed for the three enzymatichydrolysis steps.

Identification of Signature Peptides and their Parent Proteins

After peptides of interest are detected using mass spectrometry, theprotein from which a peptide originated is determined. In most instancesthis can be accomplished using a standard protocol that involvesscanning either protein or DNA databases for amino acid sequences thatwould correspond to the proteolytic fragments generated experimentally,matching the mass of all possible fragments against the experimentaldata (F. Hsieh et al., Anal. Chem. 70:1847-1852 (1998); D. Reiber etal., Anal. Chem 70:673-683 (1998)). When a DNA database is used as areference database, open reading frames are translated and the resultingputative proteins are cleaved computationally to generate the referencefragments, using the same cleavage method that was used experimentally.Likewise, when a protein database is used, proteolytic cleavage is alsoperformed computationally to generate the reference fragments. Inaddition, masses of the reference peptide fragments are adjusted asnecessary to reflect derivatizations equivalent to those made to theexperimental peptides, for example to include the exogenous affinitytag. The presence of signature peptides in the sample is detected bycomparing the masses of the experimentally generated peptides with themasses of signature peptides derived from putative proteolytic cleavageof the set of reference proteins obtained from the database. Softwareand databases suited to this purpose are readily available eitherthrough commercial mass spectrometer software or the Internet.Optionally, the peptide databases can be preselected or reduced incomplexity by removing peptides that do not contain the amino acid(s)upon which affinity selection is based.

There will, of course, be instances where peptides cannot be identifiedfrom databases or when multiple peptides in the database have the samemass. One approach to this problem is to sequence the peptide in themass spectrometer by collision induced dissociation. Ideally this isdone with a MALDI-MS/MS or ESI-MS/MS instrument. Another way to proceedis to isolate peptides and sequence them by a conventional method.Because the signature peptide strategy is based on chromatographicseparation methods, it is generally relatively easy to purify peptidesfor amino acid sequencing if sufficient material is available. Forexample, conventional PTH-based sequencing or carboxypeptidase basedC-terminal sequencing described for MALDI-MS several years ago (D.Patterson et al., Anal. Chem. 67:3971-3978 (1995)). In cases where 6-10amino acids can be sequenced from the C-terminus of a peptide, it isoften possible to synthesize DNA probes that would allow selectiveamplification of the cDNA complement along with DNA sequencing to arriveat the structure of the protein.

Internal Standard Quantification with Signature Peptides

There is a growing need to move beyond the massive effort to definegenetic and protein components of biological systems to the study of howthey and other cellular metabolites are regulated and respond tostimuli. The words “stimulus” and “stimuli” are used broadly herein andmean any agent, event, change in conditions or even the simple passageof time that may be associated with a detectable change in expression ofat least one metabolite within a cell, without limitation. For example,a stimulus can be a change in growth conditions, pH, nutrient supply, ortemperature; contact with an exogenous agent such as a drug or microbe,competition with another organism, and the like. The term “metabolite”refers, in this context, to a cellular component, preferably an organiccellular component, which can change in concentration in response to astimulus, and includes large biomolecules such as proteins,polynucleotides, carbohydrates and fats, as well as small organicmolecules such as hormones, peptides, cofactors and the like.

Accordingly, in this aspect of the invention post-biosynthetic isotopelabeling of cellular metabolites, preferably proteins and peptides, isutilized to detect cellular components that are up and/or down regulatedin comparison to control environments. Metabolites, such as proteins (orpeptides if proteolysis is employed) in control and experimental samplesare post-synthetically derivatized with distinct isotopic forms of alabeling agent and mixed before analysis. Preferably, the samples areobtained from a “biological environment,” which is to be broadlyinterpreted to include any type of biological system in which enzymaticreactions can occur, including in vitro environments, cell culture,cells at any developmental stage, whole organisms, organs, tissues,bodily fluids, and the like. As between the two samples, labeledmetabolites are chemically equivalent but isotopically distinct. In thiscontext, chemical equivalence is defined by identical chromatographicand electrophoretic behavior, such that the two metabolites cannot beseparated from each other using standard laboratory purification andseparation techniques. For example, a protein or peptide present in eachsample may, after labeling, differ in mass by a few atomic mass unitswhen the protein or peptide from one sample is compared to the sameprotein or peptide from the other sample (i.e., they are isotopicallydistinct). However, these two proteins or peptides would ideally bechemically equivalent as evidenced by their identical chromatographicbehavior and electrophoretic migration patterns.

Because >95% of cellular proteins do not change in response to astimulus, proteins (as well as other metabolites) in flux can be readilyidentified by isotope ratio changes in species resolved, for example, by2-D gel electrophoresis or 2-D chromatography. Once these proteins aredetected, they can optionally be identified using the “signaturepeptide” approach as described herein or any other convenient method.One example of how this method of the invention can be used is toanalyze patterns of protein expression in a breast cancer cell beforeand after exposure to a candidate drug. The method can also be used toanalyze changes in protein expression patterns in a cell or an organismas a result of exposure to a harmful agent. As yet another example, themethod can be used to track the changes in protein expression levels ina cell as it is exposed, over time, to changes in light, temperature,electromagnetic field, sound, humidity, and the like.

The internal standard method of quantification is based on the conceptthat the concentration of an analyte (A) in a complex mixture ofsubstances may be determined by adding a known amount of a very similar,but distinguishable substance (Λ) to the solution and determining theconcentration of A relative to Λ. Assuming that the relative molarresponse () of the detection system for these two substances is known,then[A]=[Λ]ΔThe term Δ is the relative concentration of A to that of the internalstandard Λ and is widely used in analytical chemistry for quantitativeanalysis. It is important that A and Λ are as similar as possible inchemical properties so that they will behave the same way in all thesteps of the analysis. It would be very undesirable for A and Λ toseparate. One of the best ways to assure a high level of behavioralequivalency is to isotopically label either the internal standard (Λ) orthe analyte (A).

As noted above, it is difficult to determine whether a regulatorystimulus has caused a single, or a small group of proteins in a complexmixture to increase or decrease in concentration relative to otherproteins in the sample. Determining the magnitude of this change is aneven more difficult problem. The internal standard method apparentlycannot be applied here because i) the analytes A_(1−n) undergoing changeare of unknown structure and ii) it would be difficult to selectinternal standards Λ_(1−n) of nearly identical properties.

Post-synthetic isotope labeling of proteins in accordance with themethod of the invention advantageously creates internal standards fromproteins of unknown structure and concentration. Whenever there is acontrol, or reference state, in which the concentration of proteins isat some reference level, proteins in this control state can serve asinternal standards. In a preferred embodiment, constituent peptides arelabeled after fragmentation of the proteins in the sample. The timing ofthe labeling step provides an opportunity to label every peptide in themixture by choosing a labeling method that labels at the N or the Cterminus of a polypeptide. Application of the labeling method of theinvention after the proteins have been synthesized has a furtheradvantage. Although metabolic incorporation of labeled amino acids hasbeen widely used to label proteins, it is not very reproducible and isobjectionable in human subjects. Post-sampling strategies forincorporation of labels are much more attractive.

A key advantage of the isotope labeling method of the invention is thatit detects relative change, not changes in absolute amounts of analytes.It is very difficult to determine changes in absolute amounts analytesthat are present at very low levels. This method is as sensitive tochanges in very dilute analytes as it is those that are present at greatabundance. Another important advantage of this approach is that it isnot influenced by quenching in the MALDI. This means that large numberof peptides can be analyzed irrespective of the expected quenching.

The isotope labeling method of the invention allows identification ofup- and down-regulated proteins using the affinity selection methodsdescribed above, 2-D gel electrophoresis, 1-D, 2-D or multi-dimensionalchromatography, or any combination thereof, and employs eitherautoradiography or mass spectrometry. Examples of radioisotopes andstable mass isotopes that can be used to label a metabolitepost-biosynthetically include ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³²P, ³³S,³⁴S and ³⁵S, but should be understood that the invention is in no waylimited by the choice of isotope. An isotope can be incorporated into anaffinity tag, or it can be linked to the peptide or protein in aseparate chemical or enzymatic reaction. It should be noted thataffinity selection of peptides is an optional step in the isotopelabeling method of the invention, thus the inclusion of an affinityligand in the labeling agent is optional.

In one embodiment of the isotope labeling method, proteins areisotopically labeled prior to cleavage. Proteins in a control sample arederivatized with a labeling agent that contains an isotope, whileproteins in an experimental sample are derivatized with the normallabeling agent. The samples are then combined. The derivatized proteinscan be chemically or enzymatically cleaved either before or afterseparation. Cleavage is optional; isotopically labeled proteins can, ifdesired, be analyzed directly following a fractionation step such asmultidimensional chromatography, 2-D electrophoresis or affinityfractionation. When the derivatized proteins are cleaved beforeseparation, the labeling agent preferably contains an affinity ligand,and the tagged peptide fragments are first affinity selected, thenfractionated in a 1-D or 2-D chromatography system, after which they areanalyzed using mass spectrometry (MS). In instances where thederivatized proteins are cleaved after fractionation, 2-D gelelectrophoresis is preferably used to separate the proteins. If thepeptides have also been affinity labeled, selection of theaffinity-tagged peptides can be performed either before or afterelectrophoresis. The objective of fractionation is to reduce samplecomplexity to the extent that isotope ratio analysis can be performed,using a mass spectrometer, on individual peptide pairs.

Mass spectrometric analysis can be used to determine peak intensitiesand quantitate isotope ratios in the combined sample, determine whetherthere has been a change in the concentration of a protein between twosamples, and to facilitate identification of a protein from which apeptide fragment, preferably a signature peptide, is derived.Preferably, changes in peptide concentration between the control andexperimental samples are determined by isotope ratio MALDI-massspectrometry because MALDI-MS allows the analysis of more complexpeptide mixtures, but ESI-MS may also be used when the peptide mixtureis not as complex. In a complex combined mixture, there may be hundredsto thousands of peptides, and many of them will not change inconcentration between the control and experimental samples. Thesepeptides whose levels are unchanged are used to establish the normalizedisotope ratio for peptides that were neither up nor down regulated. Allpeptides in which the isotope ratio exceeds this value are up regulated.In contrast, those in which the ratio decreases are down regulated. Adifference in relative isotope ratio of a peptide pair, compared topeptide pairs derived from proteins that did not change inconcentration, thus signals a protein whose expression level did changebetween the control and experimental samples. If the peptidecharacterized by an isotope ratio different from the normalized ratio isa signature peptide, this peptide can be used according to the method ofthe invention to identify the protein from which it was derived.

In another embodiment of the isotope labeling method of the invention,isotope labeling takes place after cleavage of the proteins in the twosamples. Derivatization of the peptide fragments is accomplished using alabeling agent that preferably contains an affinity ligand. On the otherhand, an affinity ligand can be attached to the peptides in a separatereaction, either before or after isotopic labeling. If attached afterisotopic labeling, the affinity ligand can be attached before or afterthe samples are combined. The peptide fragments in the combined mixtureare affinity selected, then optionally fractionated using a 1-D ormulti-dimensional chromatography system, or a capillary or slab gelelectrophoretic technique, after which they are analyzed using massspectrometry. In instances where the peptides are not affinity tagged,they are either affinity selected based on their inherent affinity foran immobilized ligand (preferably using IMAC or immobilized antibody orlectin) or analyzed without selection.

Alkylation with Isotopically Distinct Reagents

Proteins in control and experimental samples can be alkylated usingdifferent isotopically labeled iodoacetic acid (ICH₂COOH) subsequent toreduction. In the case of radionuclide derivatized samples, the controlis, for example, derivatized with ¹⁴C labeled iodoacetic acid and theexperimental sample with ³H labeled iodoacetate. Polypeptides thuslabeled can be resolved by 2-D gel electrophoresis, as described in moredetail below. When mass spectrometry is used in detection, normaliodoacetate can be used to derivatize the control and deuteratediodoacetate the experimental sample.

Based on the fact that proteins from control and experimental samplesare identical in all respects except the isotopic content of theiodoacetate alkylating agent, their relative molar response () isexpected to be 1. This has several important ramifications. When controland experimental samples are mixed:A=ΛΔIn this case Δ will be i) the same for all the proteins in the mixturethat do not change concentration in the experimental sample and ii) afunction of the relative sample volumes mixed. If the proteinconcentration in the two samples is the same and they are mixed in a 1/1ratio for example, then Δ=1. With a cellular extract of 20,000 proteins,Δ will probably be the same for >19,900 of the proteins in the mixture.The concentration of a regulated protein that is either up- ordown-regulated is expressed by the equation:A _(exptl.)=Λ_(contl.)Δδwhere A_(exptl) is a protein from the experimental sample that has beensynthetically labeled with a derivatizing agent, Λ_(contl.) is the sameprotein from the control sample labeled with a different isotopic formof the derivatizing agent, and δ is the relative degree of up- ordown-regulation. Because Δ is an easily determined constant derived fromthe concentration ratio of probably >95% of the proteins in a sample, δis readily calculated and proteins in regulatory flux easily identified.Isotopic Labeling of Amines

If not included as part of the alkylating agent, an isotope label can beapplied to the peptide as part of an affinity tag (if affinity selectionis contemplated), or at some other reactive site on the peptide.Although application of the internal standard isotopic label in theaffinity tag is operationally simpler and, in some cases, moredesirable, it requires that each affinity tag be synthesized in at leasttwo isotopic forms. Amine-labeling in a separate step (i.e., uncouplingthe label and the affinity ligand) is therefore a preferred alternative.

Peptides that are generated by trypsin digestion (as well as thosegenerated by many other types of cleavage reactions) have a primaryamino group at their amino-terminus in all cases except those in whichthe peptide originated from a blocked amino-terminus of a protein.Moreover, the specificity of trypsin cleavage dictates that theC-terminus of signature peptides will have either a lysine or arginine(except the C-terminal peptide from the protein). In rare cases theremay also be a lysine or arginine adjacent to the C-terminus. Primaryamino groups are easily acylated with, for example, acetylN-hydroxysuccinimide (ANHS). Thus, control samples can be acetylatedwith normal ANHS whereas experimental tryptic digests can be acylatedwith either ¹³CH₃CO—NHS or CD₃CO—NHS. Our studies show that the ε-aminogroup of all lysines can be derivatized in addition to theamino-terminus of the peptide, as expected. This is actually anadvantage in that it allows a determination of the number of lysineresidues in the peptide.

Essentially all peptides in both samples will be derivatized and hencedistinguishable from their counterparts using mass spectrometry. Thismeans that any affinity selection method or combination of affinityselection methods (other than possibly those that select for arginine orlysine, which contain free amines) can be used at any point in theprocess to obtain a selected population enriched for signature peptides.For example, isotope labeling at amines can be used to identify changesin the relative amounts of peptides selected on the basis of cysteine,tryptophan, histidine, and a wide variety of post-translationalmodifications. In this preferred embodiment of the method, isotopiclabeling and affinity labeling are two independent and distinct steps,and virtually all peptides are isotopically labeled. This providessignificantly more flexibility and greater control over the productionof signature peptides than is possible when the alkylating agent doublesas the isotope labeling agent.

Isotopic Labeling of Hydroxyls and Other Functional Groups

While acetylation is a convenient labeling method for proteins and theirconstituent peptides, other labeling methods may be useful for othertypes of cellular metabolites. For example, acetic anhydride can be usedto acetylate hydroxyl groups in the samples, and trimethylchlorosilanecan be used for less specific labeling of functional groups includinghydroxyl groups, carboxylate groups and amines.

Interpretation of the Spectra

Isotopically labeled samples (control and experimental) are mixed, thensubjected to mass spectrometry. In the case of labeled proteins (whereno proteolytic cleavage is carried out), the proteins are typicallyseparated using 2D-gel electrophoresis, multidimensional chromatography,or affinity fractionation such as immunoaffinity chromatography.Proteins from the control and experimental samples will comigrate, sinceneither isoelectric focusing (IEF), sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), nor chromatographicsystems can resolve the isotopic forms of a protein. In the case oflabeled peptides (whether or not affinity selected), peptides areoptionally subjected to fractionation (typically using reversed phasechromatography or ion exchange chromatography) prior to analysis usingmass spectrometry.

Radioisotope counting techniques can be used to discriminate between ³Hand ¹⁴C, and a mass spectrometer can readily differentiate betweendeuterated and normal species, either as proteolytic fragments or in thewhole protein when it is of low (that is, under about 15 kD) molecularweight, allowing ratios of protein abundance between the two samples tobe established. The relative abundance of most proteins will be the sameand allow Δ to be calculated. A second group of proteins will be seen inwhich the relative abundance of specific proteins is much larger in theexperimental sample. These are the up-regulated proteins. In contrast, athird group of proteins will be found in which the relative abundance ofspecific proteins is lower in the experimental sample. These are thedown-regulated proteins. The degree (δ) to which proteins are up- ordown-regulated is calculated based on the computed value of Δ.

A more detailed analysis of the interpretation of the resulting massspectra is provided using amine-labeled proteins as an example.Signature peptides of experimental samples in this example areacetylated at the amino-termini and on ε-amino groups of lysines witheither ¹³CH₃CO— or CD₃CO— residues, therefore any particular peptidewill appear in the mass spectrum as a doublet. In the simplest casewhere i) trideutero-acetic acid is used as the labeling agent, ii) theC-terminus is arginine, iii) there are no other basic amino acids in thepeptide, and iv) the control and experimental samples are mixed inexactly a 1/1 ratio before analysis, i.e., Δ=1, the spectrum shows adoublet with peaks of approximately equal height separated by 3 amu.With 1 lysine the doublet peaks were separated by 6 amu and with 2lysine by 9 amu. For each lysine that is added the difference in massbetween the experimental and control would increase an additional 3 amu.It is unlikely in practice that mixing would be achieved in exactly a1/1 ratio. Thus Δ will have to be determined for each sample and variessome between samples. Within a given sample, Δ will be the same for mostpeptides, as will also be the case in electrophoresis. Peptides thatdeviate to any extent from the average value of Δ are the ones ofinterest. The extent of this deviation is the value δ, the degree of up-or down-regulation. As indicated above, Δ will be the same for greaterthan 95% of the proteins, or signature peptides in a sample.

As noted above, amino acids with other functional groups areoccasionally labeled. In the presence of a large excess of acylatingagent hydroxyl groups of serine, threonine, tyrosine, and carbohydrateresidues in glycoconjugates and the imidazole group of histidine canalso be derivatized. This does not interfere with quantificationexperiments, but complicates interpretation of mass spectra if groupsother than primary amines are derivatized. In the case of hydroxylgroups, esters formed in the derivatization reaction are readilyhydrolyzed by hydroxylamine under basic conditions. Aclylation ofimadazole groups on the other hand occurs less frequently thanesterification and is perhaps related to amino acid sequence around thehistidine residue.

Another potential problem with the interpretation of mass spectra in theinternal standard method of the invention can occur in cases where aprotein is grossly up- or down-regulated. Under those circumstances,there will essentially be only one peak. When there is a largedown-regulation this peak will be the internal standard from thecontrol. In the case of gross up-regulation, this single peak will havecome from the experimental sample. The problem is how to know whether asingle peak is from up- or down-regulation. This is addressed by doublelabeling the control with CH₃CO—NRS and ¹³CH₃CO—NHS. Because of thelysine issue noted above, it is necessary to split the control sampleinto two lots and label them separately with CH₃CO—NHS and ¹³CH₃CO—NHS,respectively, and then remix. When this is done the control alwaysappears as a doublet separated by 1-2 amu, or 3 amu in the extreme casewhere there are two lysines in the peptide. When double labeling thecontrol with ¹²C and ¹³C acetate and the experimental sample withtrideuteroacetate, spectra would be interpreted as follows. A singlepeak in this case would be an indicator of strong up-regulation. Thepresence of the internal standard doublet alone would indicate strongdown-regulation.

Another potential problem with the double labeled internal standard ishow to interpret a doublet separated by 3 amu. Because the controlsample was labeled with CH₃CO—NHS and ¹³CH₃CO—NHS, this problem canarise only when the signature peptide has 2 lysine residues and issubstantially down-regulated to the point that there is little of thepeptide in the experimental sample. The other feature of the doubletwould be that the ratio of peak heights would be identical to the ratioin which the isotopically labeled control peptides were mixed. Thus, itmay be concluded that any time a doublet appears alone in the spectrumof a sample and Δ is roughly equivalent to that of the internal standardthat i) the two peaks came from the control sample and ii) peaks fromthe experimental sample are absent because of substantial downregulation.

Software Development

The isotope labeling method of the invention allows the identificationof the small number of proteins (peptides) in a sample that are inregulatory flux. Observations of spectra with 50 or fewer peptidesindicate that individual species generally appear in the spectra asbundles of peaks consisting of the major peptide ion followed by the ¹³Cisotope peaks. Once a peak bundle has been located, peak ratios withinthat bundle are evaluated and compared with adjacent bundles in thespectrum. Based on the isotopes used in labeling, simple rules can bearticulated for the identification of up- and down-regulated peptides inmass spectra. Software can be written that apply these rules forinterpretation.

Data processed in this way can be evaluated in several modes. One is toselect a given peptide and then locate all other peptides that are closein δ value. All peptides from the same protein should theoretically havethe same δ value (i.e., the same relative degree of up ordown-regulation). For example, when more than one protein is present inthe same 2-D gel spot there is the problem of knowing which peptidescame from the same protein. The δ values are very useful in thisrespect, and provide an additional level of selection. The same is truein 2-D chromatography. 3-D regulation maps of chromatographic retentiontime vs. peptide mass vs. δ can also be constructed. This identifiesproteins that are strongly up or down-regulated without regard to thetotal amount of protein synthesized. In some experiments, one or moregroups of proteins may be identified that have similar δ values, andidentification of the members of a group may elucidate metabolicpathways that had not previously been characterized.

The Internal Standard Method Applied to 2-D Gels

Advantageously, the internal standard method of the invention can beused in concert with conventional 2-D gel electrophoresis. The greatadvantage of 2-D electrophoresis is that it can separate severalthousand proteins and provide a very good two dimensional display of alarge number of proteins. The method of the invention allows this twodimensional display to be used to identify those species that are up- ordown-regulated. Researchers in the past have tried to do this bycomparing the staining density of proteins from different experiments(L. Anderson et al., Electrophoresis 17:443-453 (1997)); S. Pederson etal., Cell 14:179-190 (1977). However staining is not very quantitative,it is difficult to see those proteins that are present in small amounts,and multiple electrophoresis runs are required.

The detection and quantitation problems in 2-D gel electrophoresis canbe solved by post-biosynthetically derivatizing proteins with eitherradionuclides or stable isotope labeling agents before electrophoresisto facilitate detection and quantification. The great advantage of thisapproach is that the labeling agents do not have to be used in thebiological system. This circumvents the necessity of in vivoradiolabeling that is so objectionable in human studies with currentlabeling techniques. A second major advantage is that the degree of up-or down-regulation can be determined in a single analysis by usingcombinations of isotopes in the labeling agents, i.e., ¹⁴C and ³H, ¹Hand ²H, or ¹²C and ¹³C labels. Control samples are labeled with oneisotope while experimental samples are labeled with another.

Two preferred methods were described above for labeling polypeptidespost-biosynthetically: (a) labeling cysteine during alkylation andreduction of sulfhydryls and (b) labeling by acetylation of free aminogroups. Labeling through reduction and alkylation of disulfides isobviously the easiest way and the most preferred for subsequentelectrophoretic analysis because it does the least to disturb thecharge.

Radioisotopes.

Determining the ratio of radionuclides in 2-D gels requires a specialdetection method. The energy of β particles from ³H is roughly 0.018 Mevwhereas the radiation from ¹⁴C is approximately 0.15 Mev. Thisdifference in energy is the basis for discriminating between these tworadionuclides. Counting ³H requires a very thin mylar window. This factcan be exploited for differential autoradiographic detection with acommercial imager (e.g., a CYCLONE Storage Phosphor System, Packard,Meriden, Conn.). Modern imagers work by imposing a scintillator screenbetween the gel and the imager. Using a ¹⁴C control and an absorptionfilter to block ³H β radiation allows for measurement of radiationintensity for the control alone. Removing the filter and performing theautoradiographic detection again gives an intensity for ³H+¹⁴C. Usingdensitometry, it is possible to determine density ratios betweendifferent spots on the same autoradiogram and between autoradiograms.The limitation of this approach is that it is difficult to recognize i)proteins that only increase slightly in concentration, ii) up- ordown-regulation in a spot that contains multiple proteins, and iii)proteins that are substantially down-regulated. Down-regulation will berecognized by switching the isotopes, i.e., ³H is used as the controllabel and ¹⁴C as the experimental labeling agent. Once a protein spot isseen that appears to be up- or down-regulated, much better quantitationcan be achieved by excising the spot and using scintillation methods fordouble label counting.

Phosphorylation of proteins with ³²P labeled nucleotides andglycosylation in mammalian systems with ¹⁴C labeled N-acetylglucosamineare also envisioned, allowing studies of post-translationalmodifications that lend themselves to multi-isotope labeling anddetection strategies.

There are several advantages of this radioisotope version of theinternal standard as applied to 2-D gel electrophoresis. One is that itallows a large number of proteins to be screened for up- ordown-regulation from a single sample, in a single run, with a singlegel. A second is that excision of spots is not required, i.e., thedegree of manual manipulation is minimal. Yet another advantage is thatinter-run differences between gels and in the execution of the methodhave no impact on the success of the method.

Stable isotopes.

Proteins that have been reduced and alkylated with either ICH₂COOH orICD₂COOH and mixed before electrophoresis are used to produce peptidedigests in which a portion of cysteine containing peptides are deuteriumlabeled. These peptides appear as doublets separated by 2 amu in theMALDI spectrum. In those cases where there are several cysteine residuesin a peptide, the number of cysteines determines the difference in massbetween the control and experimental samples. For each cysteine, thedifference in mass increases by 2 amu. ¹³C labeling can also be used.The Δ term is derived from isotope ratios in several adjacent proteinspots on the gel whereas δ is computed from the ratio in the targetspot. Only those peptides that deviate from the average value of Δ aretargets for fisher analysis. This version of the internal standardmethod has most of the advantages of the radioisotope method in terms ofquantification, use of a single sample and gel, and reproducibility. Theradio- and stable-isotope strategies can also be combined and applied to2D gel electrophoresis. The advantage of combining them is that onlythose spots which appear to have been up- or down-regulated byradioactive analysis are subjected to MALDI-MS. When stable andradio-labeled peptides are used in the same experiment, the stableisotopes are a way to identify and fine tune quantification.

Construction of Temporal Maps

The discussion above would imply that regulation is a process that canbe understood with single measurements, i.e., after a stimulus has beenapplied to a biological system one makes a measurement to identify whathas been regulated. Single measurements at the end of the process onlyidentify the cast of characters. Regulation involves adjusting,directing, coordinating, and managing these characters. The issue inregulation is to understand how all these things occur. Regulation is atemporal process involving a cascade of events. Consider, for example,the hypothetical case in which an external stimulus might causemodification of a transcription factor, which then interacts withanother transcription factor, the two of which initiate transcription ofone or more genes, which causes translation, and finallypost-translational modification to synthesize another transcriptionfactor, etc. Temporal analysis brings a lot to understanding thisprocess. Global analysis of protein synthesis in response to a varietyof stimuli has been intensely examined and at least two mappingstrategies have been developed (R. VanBogelen et al., in F. Neidhardt etal., Ed. Escherichia coli and Salmonella: Cellular and MolecularBiology, 2nd Ed. ASM Press, Washington D.C., pp. 2067-2117); H. Zhang etal., J. Mass Spec. 31:1039-1046 (1996)).

A temporal map of protein expression can be constructed by firstidentifying all species that change in response to a stimulus, thenperforming a detailed analysis of the regulatory process during proteinflux. Identification of those proteins affected by the stimulus is mosteasily achieved by a single measurement after the regulatory event iscomplete and everything that has changed is in a new state ofregulation. Both chromatographic and electrophoretic methods can be usedto contribute to this level of understanding. The regulatory processduring protein flux is then analyzed at short time intervals andinvolves many samples. The initial identification process yieldsinformation on which species are in flux, their signature peptides, andthe chromatographic behavior of these peptides. As a result, theresearcher thus knows which samples contain specific signature peptidesand where to find them in mass spectra. Quantitating the degree to whichtheir concentration has changed with the internal standard method isstraightforward. The resulting data allows temporal maps of regulationto be constructed, and the temporal pattern of regulation will provideinformation about the pathway of response to the stimulus. The inventionthus further provides a method for developing algorithms that identifysignature peptides in regulatory change.

Microfabricated Analytical Systems

The method of the invention is amenable to automation by integratingmost of the analytical steps in a single instrument. Alkylation,reduction, proteolysis, affinity selection, and reversed phasechromatography (RPC) can be executed within a single multidimensionalchromatographic system. Samples collected from this system are manuallytransferred to MALDI plates for mass spectrometric analysis. In oneembodiment, the invention provides a single channel integrated system.In a preferred embodiment, however, the invention thus provides amicrofabricated, integrated, parallel processing, microfluidic systemthat carry out all the separation components of analysis on a singlechip.

EXAMPLES

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein

Example I Signature Peptide Approach to Detecting Proteins in ComplexMixtures

The objective of the work presented in this example was to test theconcept that tryptic peptides may be used as analytical surrogates ofthe protein from which they were derived. See Geng et al., Journal ofChromatography A, 870 (2000) 295-313; Ji et al., Journal ofChromatography B, 745 (2000) 197-210. Proteins in complex mixtures weredigested with trypsin and classes of peptide fragments selected byaffinity chromatography (in this case, lectin columns were used).Affinity selected peptide mixtures were directly transferred to ahigh-resolution reversed-phase chromatography column and furtherresolved into fractions that were collected and subjected tomatrix-assisted laser desorption ionization (MALDI) mass spectrometry.The presence of specific proteins was determined by identification ofsignature peptides in the mass spectra.

Advantages of this approach are that (i) it is easier to separatepeptides than proteins, (ii) native structure of the protein does nothave to be maintained during the analysis, (iii) structural variants donot interfere and (iv) putative proteins suggested from DNA databasescan be recognized by using a signature peptide probe.

Materials and Methods

Materials.

Human serotransferrin, human serum, N-tosyl-L-phenylalanine chloromethylketone (TPCK)-treated trypsin, concanavalin A (Con A), Bandeiraeasimplicifolia (BS-II) lectin, tris(hydroxymethyl)aminomethane (Trisbase), iodoacetic acid, tris(hydroxymethyl)aminomethane hydrochloride(Tris acid), cysteine, dithiothreitol (DTT), N-tosyl-L-lysylchloromethyl ketone (TLCK), and N-acetyl-D-glucosamine were purchasedfrom Sigma (St. Louis, Mo., USA). Nuclear extract from calf thymus wasprovided by Professor M. Bina (Department of Chemistry, PurdueUniversity, W. Lafayette, Ind., USA). LiChrospher Si 1000 (10 μm, 1000Å) was obtained from Merck (Darmstadt, Germany).3,5-Dimethoxy4-hydroxy-cinnamic acid (sinipinic acid),3-aminopropyltriethoxysilane, polyacrylic acid (PAA), and dicyclohexylcarbodiimide (DCC), d₃-C¹ acetic anhydride were purchased from Aldrich(Milwaukee, Wis., USA). Methyl-α-D-mannopyranoside was obtained fromCalbiochem (La Jolla, Calif., USA). Toluene, 4-dioxane anddimethylsulfoxide (DMSO) were purchased from Fisher Scientific (FairLawn, N.J., USA). N-Hydroxyl succinimide (NHS) and high-performanceliquid chromatography (HPLC)-grade trifluoroacetic acid (TFA) werepurchased from Pierce (Rockford, Ill., USA). HPLC-grade water andacetonitrile (ACN) were purchased from EM science (Gibbstown, N.J.,USA). All reagents used directly without further purification.

Synthesis of lectin column.

A 1-g of LiChrospher Si 1000 was activated for 5 hours at roomtemperature by addition of 40 ml 6 M HCl. The silica particles were thenfiltered and washed to neutrality with deionized water after which theywere dried initially for 2 hours at 105° C. and then at 215° C.overnight. Silica particles thus treated were reacted with 0.5%3-aminopropyltriethoxysilane in 10 ml toluene for 24 hours at 105° C. toproduce 3-aminopropylsilane derivatized silica (APS silica). Polyacrylicacid (0.503 g; M, 450 000), N-hydroxysuccinamide (1.672 g), anddicyclohexyl carbodiimide (6.0 g) were dissolved into 40 ml DMSO andshaken for 3 hours at room temperature to activate the polymer. Thereaction mixture was filtered and the activated polymer harvested in thesupernatant. Acrylate polymer was grafted to the silica particles byadding the APS silica described above to the activated acrylate polymercontaining supernatant. Following a 12-hour reaction at roomtemperature, the particles were filtered and washed sequentially with 50ml DMSO, 50 ml dioxane and 50 ml deionized water. This procedureproduces a polyacrylate coated silica with residual N-acyloxysuccinamideactivated groups, specified as NAS-PAA silica. NAS-PAA silica (0.5 g)was added to 10 ml of 0.1 M NaHCO₃ (pH 7.5) containing 0.2 Mmethyl-α-D-mannopyranoside and 200 mg Con A. The reaction was allowed toproceed with shaking for 12 hr at room temperature after whichimmobilized Con A sorbent was isolated by centrifugation and was washedwith 0.1 M Tris buffer (pH 7.5). The sorbent was stored in 0.1 M Trisbuffer (pH 7.5) with 0.2 M NaCl until use.

NAS-PAA silica (0.3 g) was added to 10 ml of 0.1 M NaHCO₃ buffer (pH7.5) containing 0.2 M N-acetyl-D-glycosamine and 20 mg BS-II lectin. Thereaction was allowed to proceed with shaking for 12 hours at roomtemperature after which the immobilized lectin containing particles wereisolated by centrifugation, washed with 0.1 M (pH 7.5) Tris buffer, andpacked into a stainless steel column (50×4.6 mm) using the wash bufferand a high-pressure pump from Shandon Southern Instruments (Sewickley,Pa., USA). Affinity columns were washed by 0.1 M Tris (pH 7.5) with 0.2M NaCl before use.

Proteolysis.

Human serotransferrin (5 mg), nuclear extract from bovine cells, orhuman serum were reduced and alkylated in the same way by adding to 1 ml0.2 M Tris buffer (pH 8.5) containing 8 M urea and 10 mM DTT. After a2-h incubated at 37° C., iodoacetic acid was added to a finalconcentration of 20 mM and incubated in darkness on ice for a further 2hours. Cysteine was then added to the reaction mixture to a finalconcentration of 40 mM and the reaction allowed to proceed at roomtemperature for 30 min. After dilution with 0.2 M Tris buffer to a finalurea concentration of 3 M, TPCK-treated trypsin (2%, w/w, of enzyme tothat of the protein) was added and incubated for 24 hours at 37° C.Digestion was stopped by adding TLCK in a slight molar excess over thatof trypsin.

Chromatography.

All chromatographic steps were performed using an Integralmicroanalytical workstation from PE Biosystems (Framingham, Mass., USA).Tryptic digested human serotransferrin (0.1 ml) was injected onto theCon A affinity column that had been equilibrated with a loading buffercontaining 1 mM CaCl₂, 1 mM MgCl₂, 0.2 M NaCl and 0.1 M Tris-HCl (pH7.5). The Con A column was eluted sequentially at 1 ml/min with twocolumn volumes of loading buffer and then 0.2 Mmethyl-α-D-mannopyranoside in 0.1 M Tris (pH 6.0). Analytes displacedfrom the affinity column with 0.2 M methyl-α-D-mannopyranoside weredirected to a 250×4.6 mm Peptide C₁₈ (PE Biosystems) analyticalreversed-phase HPLC column, which had been equilibrated for 5 minutes at1.0 ml/min with 5% ACN containing 0.1% aqueous TFA. The glycopeptideswere then eluted at 1.0 ml/min in a 35-min linear gradient to 50% ACN in0.1% aqueous TFA. Eluted peptides were monitored at 220 nm and fractionsmanually collected for matrix-assisted laser desorption ionizationtime-of-flight (MALDI-TOF) analysis.

Tryptic digested human serum (0.2 ml) was injected on the Con A andreversed-phase HPLC column using conditions similar to those used withhuman serotransferrin with the following exceptions. The reversed-phasecolumn was washed for 10 minutes at 1 ml/min with 10% ACN containing0.1% aqueous TFA and the glycopeptides were eluted at 1 ml/min with a120-min linear gradient to 70% ACN containing 0.1% aqueous TFA.

Nuclear extract (0.1 ml) was injected onto the BS-II column which hadbeen equilibrated with loading buffer, 0.2 M NaCl with 0.1 M Tris (pH7.5). After sample loading the BS-II column was washed with 20 columnvolumes of loading buffer and then eluted with 0.2 MN-acetyl-D-glycosamine in the loading buffer. Glycopeptides andglycoproteins eluted from the BS-II column were transferred to areversed-phase column, which had been equilibrated for 5 minutes at 1ml/min with 5% ACN containing 0.1% aqueous TFA. The glycoproteins werethen eluted at 1 ml/min with a 25-min linear gradient to 35% ACNcontaining 0.1% aqueous TFA. The glycopeptides were eluted at 1 ml/minwith a 35-min linear gradient to 50% ACN containing 0.1% aqueous TFA.

Synthesis of d₃-C¹ N-acetoxysuccinamide¹.

A solution of 4.0 g (34.8 mmol) of N-hydroxysuccinimide in 10.7 g (105mmol) of d₃-C¹ acetic anhydride was stirred at room temperature. After10 minutes, white crystals began to deposit. The liquid phase wasallowed to evaporate and the crystalline residue extracted with hexanewhich is allowed to dry in vacuum. The yield of the substances was 5.43g (100%), m.p. 133-134° C.

Acetylation reaction with the peptides.

A 3-fold molar excess of N-acetoxysuccinamide and d₃-C¹N-acetoxysuccinamide was added individually to the two equal aliquots of1 mg/ml peptide solution in phosphate buffer at pH 7.5, respectively.The reaction was carried at room temperature. After stirring for about4-5 hours, equal aliquots of the two samples were mixed and purified ona C₁₈ column. The collected fraction were then subjected to MALDI-MS.

MALDI-TOF-MS.

MALDI-TOF-MS was performed using a Voyager DE-RP BioSpectrometryworkstation (PE Biosystems). Samples were prepared by mixing a 1-μlaliquot with 1 μl of matrix solution. The matrix solution forglycopeptides was prepared by saturating a water-ACN (50:50, v/v), 3%TFA solution with sinipinic acid. A 1-μl sample volume was spotted intoa well of the MALDI sample plate and allowed to air-dry before beingplaced in the mass spectrometer. All peptides were analyzed in thelinear, positive ion mode by delayed extraction using an acceleratingvoltage of 20 kV unless otherwise noted. External calibration wasachieved using a standard “calibration 2” mixture from PE Biosystems.

The matrix for acetylated peptides was a solution of 3% TFA, ACN-water(50:50) solution saturated with a α-cyano-4-hydroxycinnamic acid.Peptide quantitation was performed on MALDI-TOF-MS in the reflector modeas described above. Ten spectra were collected from each sample spot andthe peak intensities averaged for each spot. A linear equation wasdeduced from the ion current intensity ratio of the deuterium-labeledand the unlabeled acetylated peptides versus the ratio of the amount ofthese two peptides.

The effect of buffer type and concentration on mass determination byMALDI-time-of-flight mass spectrometry is discussed in Amini et al.,Journal of Chromatography A, 894 (2000) 345-355.

Results and Discussion

Analytical strategy.

The work reported here is based on the proposition that signaturepeptides generated by tryptic digestion of sample proteins may beselected from complex mixtures and be used as analytical surrogates forthe protein from which they were derived. The rationale for thisapproach is that (i) it will be easier to separate and identifysignature peptides than intact proteins in many cases, (ii) therequisite isolation of proteins for reagent preparation andidentification can be precluded by synthesizing signature peptidesidentified in protein and DNA databases, and (iii) it is easier totryptic digest all proteins in a single reaction than to isolate anddigest each individually as in the 2D electrophoretic approach.

A five-step protocol was used for production of signature peptides. Thefirst step was to select a sample from a particular compartment oforganelle. Simple methods, such as centrifugal fractionation oforganelles, greatly enrich a sample in the components being examined.The second step embodied reduction and alkylation of all proteins in thesample. In some cases the alkylating agent can be affinity labeled tofacilitate subsequent selection of cysteine-containing peptides. Thethird step was tryptic digestion of all polypeptides in the reduced andalkylated sample. A few to more than a hundred peptides will begenerated from each protein, depending on solubility and ease ofdigestion. Although data are not presented, it was found that trypsinwill partially digest leather and by so doing generates signaturepeptides. This potentially offers an avenue to the analysis of insolubleproteins. The enormous complexity of the sample produced by proteolysiswas reduced in a third step by using affinity chromatography methods toselect peptides with unique structural features. Affinity selectedpeptides were then fractionated by high-resolution RPLC in a fourthstep. And finally, target peptides from RPLC fractions were identifiedby MALDI-TOF-MS mass in the fifth step.

The analytical strategy employed in this study focused on the ability ofCon A lectin columns to select glycopeptides from tryptic digests, RPLCto further fractionate the selected peptides, and MALDI-TOF-MS toidentify specific peptides in RPLC fractions. Lectin columns have beenwidely used to purify glycopeptides, generally for the purpose ofstudying the oligosaccharide portion of the conjugate. Whencharacterization of the sugar moiety is the object, it is important tofractionate as many of the glycoforms as possible, either with seriallectin columns, anion-exchange chromatography, or capillaryelectrophoresis. The focus of this work, in contrast, was on the peptideportion of the glycoconjugate. Any glycoform containing the signaturepeptide backbone is appropriate for protein identification. Con A hashigh affinity for N-type hybrid and high-mannose oligosaccharides,slightly lower affinity for complex di-antenary oligosaccharides, andvirtually no affinity for complex N-type tri- and tetra-antenaryoligosaccharides. Most of the N-type glycoproteins contain glycoformsthat are recognized by Con A. Thus, a Con A column is ideal forselecting glycopeptides from digests of N-type glycoproteins.

Compartmentalization.

Protein(s) of interest often residue in a particular compartment in acell or organism. The act of first isolating the compartment withinwhich the protein is contained can produce a very substantialsimplification of the sample. One system chosen for this study wasglycoproteins in bovine cellular nuclei.

Glycoproteins in the nuclei of mammalian cells are uniquely different tothose found in the cytosol. Higher animal cells reversibly O-glycosylatesome nuclear proteins with a single N-acetyl glucosamine (O-GlcNAc) at aspecific serine or threonine residue. It is thought that this O-GlcNAcglycosylation is associated with transcription factors and is part of acontrol process; thus it is necessary to have enzymes for bothglycosylate and deglycosylate in the same compartment. It was anobjective in this study to gain a rough idea of the number of theseglycoproteins in the nuclei of bovine pancreas cells.

Subsequent to the isolation of nuclei by centrifugation, histones wereselectively removed and O-glycosylated proteins isolated as a group bychromatography on a Bandeiraea simplicifolia (BS-II) lectin affinitycolumn. This lectin is specific for N-acetyl glucosamine. A silica basedBS-II column was synthesized and coupled with a switching valve to areversed-phase column. This two-dimensional chromatographic system wasused to concentrate and purify glycoproteins from nuclei. Reversed-phasechromatography (FIG. 2) and 2D gel electrophoresis of the proteinfraction eluted from the lectin column by N-acetyl-D-glucosamine (0.20M) confirm the presence of some 25-35 major components in the sample.More components may be present but below the limits of detection.Considering that some 20,000 proteins may be expressed in mammaliancells, this is much simpler than anticipated. The results of this studyshow that compartmentalization and affinity selection of specificproteins from a cell can greatly reduce the number of proteins in asample.

When the protein sample used for glycoprotein analysis was reduced,alkylated with iodoacetamide, and trypsin digested before chromatographyon the (BS-II) lectin affinity column, the reversed-phase chromatogramof the glycopeptides captured by the affinity column again showsunexpected simplicity (FIG. 3). Mass spectra of selected peaks (FIG. 4)indicate a relatively low degree of complexity in fractions collectedfrom the reversed-phase column. No attempt was made to identify thesepeptides by either database searches or multidimensional MS.

Signature peptide selection from serotransferrin.

Serotransferrin, i.e., transferrin from serum, was chosen as a modelprotein to examine affinity selection of affinity peptides. Humanserotransferrin is a glycoprotein of M, 80,000 containing 679 amino acidresidues. Potential sites for N-glycosylation are found in the sequenceat residues Asn₄₁₃ and Asn₆₁₁. The reversed-phase chromatogram of atryptic digest (FIG. 5 a) is seen to be substantially reduced incomplexity when non-glycosylated peptides are first removed with aconcanavalin A affinity chromatography column (FIG. 5 b). The peptidesglycosylated at residues Asn₄₁₃ and Asn₆₁₁ eluted at 27.5 and 33.4% ofsolvent B, respectively. MALDI-MS of the two major components from FIG.6 b are seen in FIGS. 6 a and 6 b, respectively. Although thechromatographic peaks appear to be homogenous, MALDI-TOF-MS indicatesconsiderable heterogeneity within the two fractions. This is asexpected. It is known that there is often substantial heterogeneity inthe oligosaccharide portion of a glycopeptide. The stationary phase ofthe reversed-phase column interacts almost exclusively with the peptideregion of glycopeptides, essentially ignoring the oligosaccharideportion. This means that glycopeptides which are polymorphic in theoligosaccharide part of the molecule will produce a singlechromatographic peak, albeit slightly broader than that of a singlespecies. On the other hand, MALDI-TOF-MS discriminates on the basis ofmass and detects all species that differ in mass without regard tostructure. Used together, these two methods produce a high degree ofstructural selectivity.

Identification of serotransferrin signature peptides from serum.

Based on the solvent composition known to elute the serotransferringlycopeptides and their mass spectra, an experiment was undertaken toidentify these signature peptides in a tryptic digest of human serumproteins. Chromatograms in FIGS. 7 a and 7 b show the enormouscomplexity of the glycopeptide mixture selected from a tryptic digest ofhuman serum by a Con A affinity chromatography column. Fractions elutingbetween 27 and 28% and between 33 and 34% were collected from thereversed-phase column and their mass spectra compared with that of humanserotransferrin. Although extremely complex, mass spectra (FIGS. 6 a and6 b) obtained from fractions corresponding in chromatographic propertiesto the serotransferrin glycopeptides reveal the presence of thesesignature peptides in the serum sample. FIG. 8 a shows masses at 3861,4163 and 4213 u, matching the glycopeptide peaks from FIG. 6 a. Masserror was typically <4 u using external calibration. Because of therelatively lower amount of the human transferrin in an individual'sserum, higher laser power was used to generate the spectra than that inpure human transferrin. Therefore, peak intensity were lower andspectral resolution were lower. In order to increase signal to noiseratio, all the spectra were smoothed by a 19-point averaging process.This caused the mass error to be a little higher. Glycoforms at 3459,3614 and 3895 u were either absent or ion suppressed sufficiently sothat they could not be seen. We also checked the fraction from 25 to 27%and from 29 to 31%, there was no more than one peak matchingglycopeptide peaks from FIG. 6 a. It demonstrated that the matching ofthese peaks were not coincident. FIG. 8 b shows that 4595. 4634, 4710and 4753 matched the glycopeptides peaks from FIG. 6 b. Again, fractionsfrom 31 to 33% and 34 to 36% were checked and no matching was found. Thefact that the spectra are not identical in relative intensities to thestandards can be explained by possible reasons: differences inglycosylation ratio between the reference protein and that in the serumsample of an individual; inter-run variations in MALDI spectra resultingfrom difference in MALDI ionization.

Although not examined, other modes of selection are also potentiallypossible. A variety of Iectins are available that allow the selection ofspecific types of post-translational modification on the basis ofoligosaccharide structure. Antibodies would be another way to select forspecific types of post-translational modification such asphosphorylation. Antibodies have also been used to select dinitrophenylderivatized amino acids, such as tryptophan. Alkylation of cysteine witha biotinylated form of maleimide has been suggested as another way toselect cysteine-containing peptides with avidin. Perhaps doubleselection by a combination of these affinity methods will give evenhigher degrees of selectivity.

It is concluded that signature peptides derived from tryptic digests ofcomplex protein mixtures can be used as analytical surrogates, at leastin the case of glycoproteins. Even in the case of samples with thecomplexity of human serum, the multidimensional analytical approach ofaffinity chromatography, reversed-phase chromatography and massspectrometry has sufficient resolution to identify single signaturepeptide species. Because the whole protein is not needed for analysis,this strategy is particularly suited to the identification of proteinsof limited solubility or that are suggested from DNA data bases but havenever been isolated.

Example II Sample Protocol for Analysis of Protein Mixtures

The following protocol is one of many according to the invention thatare useful for analyzing complex protein mixtures.

Step 1.

Reduction of entire sample containing several thousand proteins in arobotic sample handling system.

Step 2.

Alkylate sulfhydryl groups. If cysteine selection is desired thealkylating reagent is an affinity tagged maleimide. If the selectionwill be for another amino acid, the alkylating agent will be iodoaceticacid or iodoacetamide.

Step 2′.

If another amino acid is to be affinity selected, such as tyrosine, thatderivatizing agent is added at this time.

Step 3.

Proteolysis; generally with trypsin, but any proteolytic enzyme orcombination of enzymes could be used. Enzymatic digest could either bedone in the robotic system or with an immobilized enzyme column.

Step 4.

An affinity sorbent is used to adsorb affinity tagged species.Non-tagged peptide species are eluted to waste.

Step 5.

Tagged species are desorbed from the affinity sorbent.

Step 6.

Tagged species are chromatographically resolved. In the simplest casethe sample is subjected to high resolution reverse phase chromatography(RPC) only. Still higher resolution can be achieved by using twodimensional chromatography. Step gradient elution ion exchangechromatography with RPC of each fraction is a good choice. Given thatthe ion exchange column could split the tagged species into 50 fractionsand the RPC column had a peak capacity of 200, it is possible togenerate 10,000 fractions for MALDI. It is estimated that the totalnumber of sulfhydryl containing peptides would not exceed 20,000. Thiswould mean that no sample would contain more than 2-10 peptides. MALDIshould be very capable of handling 1-30 peptides per sample.

Step 7.

Samples are collected from the chromatographic system and transferreddirectly to the MALDI plates. Alternatively, if the sample is not toocomplex, analytes are electrosprayed directly into an ESI-MS.

Example III Representative Amino Acid Derivatizations

1. Tryptophan can be derivatized with 2,4-dinitrophenylsulfenylchloride. (Biochem. Biophys. Acta. 278, 1 (1972)]. Reaction conditions:50% acetic acid, 1 hour, room temperature. Selection is based ondinitrophenyl-directed antibodies.

2. Cysteine can be derivatized with an affinity tagged maleimide. Normaland deuterium labeled tags are mixed so that tagged species are easilyidentified in the MALDI spectrum as a doublet that is three mass unitsapart.

For example, cysteine residue in a polypeptide can be derivatized withaffinity tagged D₂-maleimide. Here, the affinity tag is peptide R₅-R₇.

3. Cysteine can alternatively be derivatized with 2,4-dinitrobenzylchloride. Conditions: pH 5, 1 hour, room temperature.

4. Methionine can be derivatized under acidic conditions. Thisderivatizing agent also derivatizes histidine at pH 5. The substantialionization of histidine at pH 3 apparently diminishes its alkylation. Inview of the fact that histidine reacts with this reagent, it ispreferable to remove histidine peptides with IMAC before derivatization.

Example IV Advantages and Disadvantages of Selective Capture of SpecificAmino Acids

1. Cysteine

a. Biotinylation of maleimide.

-   -   Positives—very high affinity capture. Avidin columns are readily        available.    -   Negatives—it takes very acidic conditions to release from        columns. A large molecule (avidin) is being used to capture a        small molecule, thus a large column is needed to capture enough        peptide for analysis.

b. Histidine labeling of maleimide.

-   -   Positives—very simple columns may be used that are of high        capacity.    -   Negatives—non-cysteine containing peptides in the digest that        also contain histidine will also be selected. In addition, the        mass starts to get a little high.

c. Peptide labeling and antibody (Ab) capture.

-   -   Positives—very high capture efficiency. Easy to release captured        peptide.    -   Negatives—a large molecule (Ab) is being used to capture a small        molecule, thus a large and expensive column is needed to capture        enough peptide for analysis.

d. Dinitrophenylation.

-   -   Positives—very simple organic chemistry. Antibody capture is        very efficient.    -   Negatives—a large molecule (Ab) is being used to capture a small        molecule, thus a large and expensive column is needed to capture        enough peptide for analysis. It is also difficult to heavy        isotope label 2,4-DNP.        2. Tryptophan

a. Dinitrophenylation.

-   -   Positives—very simple organic chemistry. Antibody capture is        very efficient.    -   Negatives—a large molecule (Ab) is being used to capture a small        molecule, thus a large and expensive column is needed to capture        enough peptide for analysis. It is also difficult to heavy        isotope label 2,4-DNP.        3. Methionine

a. Dinitrophenylation.

-   -   Positives—very simple organic chemistry. Antibody capture is        very efficient.    -   Negatives—a large molecule (Ab) is being used to capture a small        molecule, thus a large and expensive column will be needed to        capture enough peptide for analysis. It is also difficult to        heavy isotope label 2,4-DNP.

b. Histidine labeling.

-   -   Positives—very simple columns may be used that are of high        capacity.    -   Negatives—non-cysteine containing peptides in the digest that        also contain histidine will also be selected. In addition, the        mass starts to get a little high.

c. Peptide labeling and antibody capture.

-   -   Positives—very high capture efficiency. Easy to release captured        peptide.    -   Negatives—a large molecule (Ab) is being used to capture a small        molecule, thus a large and expensive column is needed to capture        enough peptide for analysis.

d. Biotinylation.

-   -   Positives—very high affinity capture. Avidin columns are readily        available.    -   Negatives—it takes very acidic conditions to release from        columns. A large molecule (avidin) is being used to capture a        small molecule, thus a large column is needed to obtain enough        peptide for analysis.        4. Tyrosine

a. Nitrophenylation and antibody capture.

-   -   Positives—very simple organic chemistry. Antibody capture is        very efficient.    -   Negatives—a large molecule (Ab) is being used to capture a small        molecule, thus a large and expensive column is needed to capture        enough peptide for analysis. It is also difficult to heavy        isotope label NP.

b. Reaction with diazonium salts to form wide variety of derivatives.

-   -   Positives—simple reaction that is well known.    -   Negatives—very hydrophobic group, affinity tag must be attached,        cross reacts with other amino acids.        5. Histidine

a. Capture with an IMAC column.

Example V Simple Post-Digestion Secondary Labeling Protocol

*Affinity labeling cysteine residues is optional. It should be noted,however, that cysteine must be alkylated at this point and if it is notaffinity labeled during reduction, it can never be labeled.

Example VI Sample Pre-Digestion Labeling Protocol

*Affinity labeling cysteine residues in this case is optional. It shouldbe noted, however, that cysteine must be alkylated at this point and ifit is not affinity labeled during reduction it can never be labeled.

Example VII Isotopically Labeled Internal Standard Quantification

One of the issues with the signature peptide approach is how toquantitate the protein being identified. Because tryptic digests ofsamples containing many proteins are enormously complex, the mixturegenerally will not be resolved into individual components byreversed-phase chromatography. Simple absorbance monitoring isprecluded. This will even be true with affinity selected samples as wasseen in FIGS. 3 and 7. FIGS. 7 a and 7 b shows that there can be so manycomponents in reversed-phase chromatograms of affinity selected samplesthat quantification of any particular peptide is impossible. The nextavenue to quantification would be to use peak height in the MALDI-TOFspectrum. Unfortunately, MALDI-TOF is not very quantitative. A bettermethod is needed.

Internal standards are frequently used in quantitation. The internalstandard method of quantification is based on the concept that theconcentration of an analyte in a complex mixture of substances may bedetermined by adding a known amount of a very similar, butdistinguishable substance to the solution and determining theconcentration of analyte relative to a known concentration of theinternal standard. Assuming that the relative molar response of thedetection system for these two substances (/R) can be determined, thenA=Λ[/R]Δ. The term A is the instrument response to analyte, Λ isinstrument response to the internal standard, R is specific molarresponse to analyte, is specific molar response to the internalstandard, and Δ is the relative concentration of analyte to that of theinternal standard. It is important that these substances are as similaras possible in chemical properties so they will behave the same way inall the steps of the analysis. In view of the fact that the last step ofthe analytical protocol used to identify signature peptides is MS,isotopic labeling of either the internal standard or the analyte wouldbe the best way to produce an internal standard. Chromatographic systemsare generally not able to resolve isotopic forms of an analyte whereasisotopically labeled species are easily resolved by MS. Behavioralequivalency in all stages except MS is critical. The question is how toeasily create isotopically labeled internal standards of peptides inmixtures.

This may be done in two ways. One is through the synthesis of peptidesin which one of the amino acids is labeled. The second is byderivatizing peptides with an isotopically labeled reagent. Although itis more lengthy, the second route was chosen because it can also be usedto create internal standards of unknown structures. This is critical inproteomic studies where the object is to identify unknown proteins inregulatory flux.

Data are presented that suggest proteins may indeed be quantified astheir signature peptides by using isotopically labeled internalstandards. Signature peptides generated by trypsin digestion have aprimary amino group at their amino-terminus in all cases except those inwhich the peptide originated from the blocked amino-terminus of aprotein. The specificity of trypsin cleavage dictates that theC-terminus of signature peptides will have either a lysine or arginine(except the C-terminal peptide from the protein) and that in rare casesthere may also be a lysine or arginine adjacent to the C-terminals.Primary amino groups of peptides were acylated withN-hydroxysuccinimide.

When analyzed by MALDI-MS in the positive ion mode, it is seen (FIG. 9)that a peptide with five amino groups (KNNQKSEPLIGRKKT; SEQ ID NO:1) canbe quantitatively derivatized with this reaction. Internal standardpeptides are acetylated with the trideuteroacetylated analogue ofN-hydroxysuccinimide. This means that peptides in samples containingboth the native and deuterated internal standard species (FLSYK; SEQ IDNO:2) would appear in the mass spectrum as a doublet (FIG. 10 a). Thepresence of a carboxyl group in all tryptic peptides allows them to beanalyzed by MALDI-TOF-MS in the negative ion mode. It was found that theε-amino group of all lysines can be derivatized in addition to theamino-terminus of the peptide, as expected. Arginine residues are notacetylated. This means that 3 amu would be added for each lysine whenusing trideutero-N-hydroxysuccinimide. The number of lysines in apeptide is revealed by the mass shift. (Multiple basic amino acidsoccasionally occur at the C-terminus with trypsin.) It is also possibleto differentiate between peptides in which the only basic amino acid islysine, or arginine, or a combination of the two. Peptides in which theonly basic amino acid is lysine have no positive charge afteracetylation. No spectra will be produced in the positive ion mode of ionacceleration unless a cationizing agent is added to the peptide.Actually, the peptide in this case picks up sodium and potassium ionsfrom the matrix in the MALDI source, causing an increase in massequivalent to that of sodium or potassium. Because the mass of these twoions is different, they appear in the spectrum as a double. When coupledwith the fact that the lysine peptide described above in FIG. 10 a isalso deuterated, the mass spectrum of this peptide in the positive ionmode of acceleration will show four peaks (FIG. 10 b).

The mass spectrum for any peptide in a sample containing an isotopicallylabeled internal standard will appear as at least a doublet. Thesimplest case would be the one where (i) trideutero-NAS was used as thelabeling agent, (ii) the C-terminus was arginine, and (iii) there wereno other basic amino acids in the peptide. Spectra in this case show adoublet in which the two peaks are separated by 3 u (FIG. 11 b). Withone lysine the doublet peaks were separated by 6 u (FIG. 11 a) and withtwo lysine by 9 u. For each lysine that is added the difference in massbetween the experimental and control would increase an additional 3 u.Quantification of the relative amounts of both lysine and argininecontaining peptides using MALDI-TOF and isotopically labeled internalstandards was studied. A linear equation was deduced from the ioncurrent intensity ratio of deuterium-labeled and unlabeled acetylatedpeptides versus the known ratio of the amount of these two peptides. Theequation of the arginine-containing peptide (TAGFLR; SEQ ID NO:3) wasy=0.9509x−0.3148 (R²=0.9846) while that for a lysine-containing peptide(FLSYK; SEQ ID NO:2) was y=0.9492x+0.4112 (R²=0.9937). The term y standsfor the intensity ratio of the deuterium-labeled to unlabeled acetylatedpeptides and x stands for the relative amount of these two peptides.

These results strongly suggest that a method in which internal standardpeptides are created by isotopic labeling and ratios of native tointernal standard species quantified by MS will be useful in determiningthe relative concentration of signature peptides.

It is concluded that isotopically labeled internal standard analysisprovides a useful method for the quantification of peptides. There is astrong possibility that when coupled with signature peptide derived fromproteins, these combined methods will provide a powerful new method forthe quantification of multiple proteins in complex mixtures.

Example VIII Sample Protocol for Analysis of Protein Expression

The following protocol is one of many according to the invention thatare useful for analyzing protein expression levels.

Step 1.

Reduction of control and experimental samples containing severalthousand proteins in robotic sample handling system.

Step 2.

Alkylate sulfhydryl groups in experimental sample. If cysteine selectionis desired the alkylating reagent is an affinity tagged maleimide. Ifthe selection will be for another amino acid, the alkylating agent isiodoacetic acid or iodoacetamide.

Step 2′.

Alkylate sulfhydryl groups in the control sample. If cysteine selectionis desired, the alkylating reagent is a heavy isotope affinity taggedmaleimide. If the selection will be for another amino acid, thealkylating agent is heavy isotope labeled iodoacetic acid oriodoacetamide. This allows proteins originating from the experimentalsample to be distinguished from those originating from the controlsample.

Step 3.

The experimental and isotopically labeled control samples are combined.

Step 4.

The proteins are separated by 2-D electrophoresis or 2-D chromatography.Reduction and alkylation may destroy tertiary and quaternary structureof the proteins. This would have a large impact on electrophoresis andchromatography, but the results could still be extrapolated to thenative protein sample.

Step 5.

Purified or partially purified proteins are subjected to proteolysis;generally with trypsin, but any proteolytic enzyme or combination ofenzymes could be used. Enzymatic digest would either be done in arobotic system or with an immobilized enzyme column.

Step 6.

Digested samples are transferred directly to the MALDI plates.

Example IX Use of Fragment Ions to Distinguish Isobaric Peptides

A C-terminal arginine containing peptide (NH₂-H-L-G-L-A-R-OH; 1 mg) (SEQID NO:4) was dissolved in 1 ml of 0.1M phosphate buffer pH 7.5. Thissolution was then divided into two equal parts (500 ul each). One partwas acetylated with N-(¹H₃)acetoxysuccinimide and the other was withN-(²H₃)acetoxysuccinimide. Both parts were then mixed and purified on aC18-reversed phase column (RPC). Fractions from the RPC were collectedand subjected to ESI-MS/MS. The singly charged precursor ion isotopecluster of m/z 708.50/711.50 [M+H] was isolated and subjected tocollision-activated dissociation (CAD).

The tandem mass spectrum given by the CAD of singly chargeddifferentially acetylated precursor ion isotope cluster of Ac-HLGLAR-OH(m/z 708.50/711.50) (SEQ ID NO:4) yields fragment ions listed inTable 1. Both N- and C-terminal fragment ions of type a, b and y arepresent in this spectrum. Complete b_(n) or y_(n) ion series are notseen in this spectrum. All prominent N-terminal fragment ions (a and btype) appeared as isotope clusters, separated by 3 amu. In contrast, allC-terminal (y-type) fragment ions are not seen as isotope clustersseparated by 3 amu; rather they coincide, since these ions do notcontain an acetyl group. Isotope ratios of all b-ions were determined bythe peak heights of acetylated form divided by the peak heights oftrideuteroacetylated form. For example relative abundance (peak height)of m/z 534.1 divided by the relative abundance of m/z 537.2 was used toget the ratio 1.07 of b5 ion (see Tables 1 and 2). Fragment ions y5-y2confirms the N-terminal sequence of Ac-H-L-G-L (SEQ ID NO:5), whereasfragment ions b5-b2 confirms the C-terminal sequence of G-L-A-R-OH (SEQID NO:6).

It is evident that the isotope labeling ratios carry through from theprecursor ion to the fragment ions. This differential labeling can beused to achieve relative quantification of peptides by tandem massspectrometry in proteomics. This also permits multiple precursor ionshaving the same mass (“isobaric peptides”) to be readily distinguishedand quantified after CAD of the parent ion in this second massspectrometry dimension.

TABLE 1 Fragment ions assignments m/z of m/z of m/z of m/z of¹H₃-acetylation ²H₃-acetylation Assignments ¹H₃-acetylation²H₃-acetylation Assignments 691.3 694.3 M-NH₃ 673.3 676.3 M-H₂O-17 690.3693.3 M-H₂O 648.3 651.3 M-H₂O-Ac 552.2 555.1 b5 + H₂O 529.4 529.4 y5534.1 537.2 b5 512.2 512.2 y5-NH₃ 463.1 466.1 b4 416.3 416.3 y4 350.0353.1 b3 399.3 399.3 y4-NH₃ 292.9 296.0 b2 359.3 359.3 y3 435.1 438.1 a4246.3 246.3 y2

TABLE 2 Statistical analysis of fragment ion ratios of differentiallyacetylated peptide NH₂-H-L-G-L-A-R-OH (SEQ ID NO:4) ExperimentalExpected Fragment ions ratio Mean +/− SD ratio % Error M-NH₃ 9.6/9.0 =1.07 1.0 M-H₂O 7.54/7.5 = 1.0 1.0 M-H₂O-17 0.64/0.61 = 1.05 1.0 M-H₂O-Ac7.97/7.61 = 1.05 1.0 b5 + H₂O 2.4/2.3 = 1.04 1.08 ±± 0.060 1.0 8.0 b58.5/7.97 = 1.07 1.0 b4 8.68/8.1 = 1.07 1.0 b3 4.6/4.1 = 1.12 1.0 b21.44/1.2 = 1.20 1.0 a4 2.65/2.29 = 1.16 1.0

The complete disclosures of all patents, patent applications includingprovisional patent applications, and publications, and electronicallyavailable material cited herein are incorporated by reference. Theforegoing detailed description and examples have been provided forclarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described; many variations will be apparent to one skilled inthe art and are intended to be included within the invention defined bythe claims.

1. A method for detecting a difference in the concentration of a proteinpresent in a first sample and in a second sample, each sample comprisinga plurality of proteins, the method comprising: covalently attaching afirst isotopic variant of a chemical moiety to a protein in the firstsample to yield at least one first isotopically labeled protein;covalently attaching a second isotopic variant of the chemical moiety toa protein in the second sample to yield at least one second isotopicallylabeled protein, wherein the first and second isotopically labeledproteins are chemically equivalent yet isotopically distinct; mixing atleast portions of the first and second samples to yield a combinedsample; and subjecting the combined sample to mass spectrometricanalysis to determine a normalized isotope ratio characterizing proteinswhose concentration is the same in the first and second samples and anisotope ratio of the first and second isotopically labeled proteins,wherein a difference in the isotope ratio of the first and secondisotopically labeled proteins and the normalized isotope ratio isindicative of a difference in concentration of the protein in the firstand second samples.
 2. The method of claim 1 further comprisingfractionating the combined sample to yield at least one fractioncomprising the isotopically labeled first and second proteins prior todetermining the isotope ratios.
 3. The method of claim 2 whereinfractionating the combined sample comprises subjecting the proteins tomultidimensional chromatography, two-dimensional electrophoresis,affinity fractionation, or a combination thereof.
 4. A method fordetecting a difference in the concentration of a protein present in afirst sample and in a second sample, each sample comprising a pluralityof proteins, the method comprising: covalently attaching a firstisotopic variant of a chemical moiety to a protein in the first sampleto yield at least one first isotopically labeled protein; covalentlyattaching a second isotopic variant of the chemical moiety to a proteinin the second sample to yield at least one second isotopically labeledprotein, wherein the first and second isotopically labeled proteins arechemically equivalent yet isotopically distinct; fragmenting proteins inthe first and second samples to yield first and second isotopicallylabeled peptides in the first and second samples, respectively; mixingat least portions of the first and second samples to yield a combinedsample, wherein mixing is performed before or after fragmentation; andsubjecting the combined sample to mass spectrometric analysis todetermine a normalized isotope ratio characterizing peptides derivedfrom proteins whose concentration is the same in the first and secondsamples and an isotope ratio of the first and second isotopicallylabeled peptides, wherein a difference in the isotope ratio of the firstand second isotopically labeled peptides and the normalized isotoperatio is indicative of a difference in concentration in the first andsecond samples of a protein derived from the peptide.
 5. A method fordetecting a difference in the concentration of a protein present in afirst sample and in a second sample, each sample comprising a pluralityof proteins, the method comprising: fragmenting proteins in the firstand second samples to yield at least one peptide in each sample;covalently attaching a first isotopic variant of a chemical moiety to apeptide in the first sample to yield at least one first isotopicallylabeled peptide; covalently attaching a second isotopic variant of thechemical moiety to a peptide in the second sample to yield at least onesecond isotopically labeled peptide, wherein the first and secondisotopically labeled peptides are chemically equivalent yet isotopicallydistinct; mixing at least portions of the first and second samples toyield a combined sample; and subjecting the combined sample to massspectrometric analysis to determine a normalized isotope ratiocharacterizing peptides derived from proteins whose concentration is thesame in the first and second samples and an isotope ratio of the firstand second isotopically labeled peptides, wherein a difference in theisotope ratio of the first and second isotopically labeled peptides andthe normalized isotope ratio is indicative of a difference inconcentration in the first and second samples of a protein derived fromthe peptide.
 6. A method for detecting a difference in the concentrationof a protein originally present in a first sample and in a secondsample, each sample comprising a plurality of peptides derived fromfragmentation of proteins originally present in the sample, the methodcomprising: covalently attaching a first isotopic variant of a chemicalmoiety to a peptide in the first sample to yield at least one firstisotopically labeled peptide; covalently attaching a second isotopicvariant of the chemical moiety to a peptide in the second sample toyield at least one second isotopically labeled peptide, wherein thefirst and second isotopically labeled peptides are chemically equivalentyet isotopically distinct; mixing at least portions of the first andsecond samples to yield a combined sample; and subjecting the combinedsample to mass spectrometric analysis to determine a normalized isotoperatio characterizing peptides derived from proteins whose concentrationis the same in the first and second samples and an isotope ratio of thefirst and second isotopically labeled peptides, wherein a difference inthe isotope ratio of the first and second isotopically labeled peptidesand the normalized isotope ratio is indicative of a difference inconcentration in the first and second samples of a protein derived fromthe peptide.
 7. The method of claim 6 wherein the first and secondchemical moieties are attached to at least one amino group on peptidesin the first and second samples.
 8. The method of claim 6 wherein eachmember of at least one pair of chemically equivalent, isotopicallydistinct peptides comprises at least one affinity ligand, the methodfurther comprising, prior to determining the isotope ratios, contactingthe peptides with a capture moiety to select peptides comprising the atleast one affinity ligand.
 9. The method of claim 8 further comprisingsubjecting the selected peptides comprising the at least one affinityligand to mass spectrometric analysis to detect at least one peptide;and identifying the protein from which the detected peptide was derived.10. The method of claim 9 wherein the detected peptide is a signaturepeptide for a protein, the method further comprising determining themass of the signature peptide and using the mass of the signaturepeptide to identify the protein from which the detected peptide wasderived.
 11. The method of claim 10 further comprising determining theamino acid sequence of the detected peptide and using the amino acidsequence of the detected peptide to identify the protein from which thedetected peptide was derived.
 12. The method of claim 8 furthercomprising subjecting the selected peptides comprising the at least oneaffinity ligand to mass spectrometric analysis to determine peakintensities; and quantitating isotope ratios from the peak intensities.13. The method of claim 8 further comprising, prior to contacting thepeptides with the capture moiety, covalently attaching at least oneaffinity ligand to at least one peptide derived from the fragmentationof the proteins.
 14. The method of claim 5 further comprising, prior tofragmenting the proteins, covalently attaching at least one affinityligand to at least one protein in the sample.
 15. The method of claim 5further comprising reducing and alkylating the proteins with analkylating agent prior to fragmenting the proteins.
 16. The method ofclaim 15 wherein the at least one affinity ligand is covalently attachedto the alkylating agent.
 17. The method of claim 8 wherein the at leastone affinity ligand is covalently attached to an amino acid of thepeptide selected from the group consisting of cysteine, tyrosine,tryptophan, histidine and methionine.
 18. The method of claim 8 whereinthe affinity ligand comprises a moiety selected from the groupconsisting of a peptide antigen, a polyhistidine, a biotin, adinitrophenol, an oligonucleotide and a peptide nucleic acid.
 19. Themethod of claim 8 wherein at least one peptide comprises an endogenousaffinity ligand.
 20. The method of claim 19 wherein the endogenousaffinity ligand comprises a moiety selected from the group consisting ofa cysteine, a histidine, a phosphate group, a carbohydrate moiety and anantigenic amino acid sequence.
 21. The method of claim 10 comprisingattaching a plurality of affinity ligands, each to at least one proteinor peptide, and contacting the peptides with a plurality of capturemoieties to select peptides comprising at least one affinity ligand. 22.The method of claim 5 wherein the proteins are fragmented using anenzyme selected from the group consisting of trypsin, chymotrypsin,gluc-C, endo lys-C, pepsin, papain, proteinase K, carboxypeptidase,calpain and subtilisin.
 23. The method of claim 6 further comprisingfractionating the peptides prior to determining the isotope ratios. 24.The method of claim 23 wherein fractionating the peptides comprisessubjecting the peptides to at least one separation technique selectedfrom the group consisting of reversed phase chromatography, ion exchangechromatography, hydrophobic interaction chromatography and sizeexclusion chromatography, capillary gel electrophoresis, capillary zoneelectrophoresis, and capillary electrochromatography, capillaryisoelectric focusing, immobilized metal affinity chromatography andaffinity electrophoresis.
 25. The method of claim 6 wherein the samplecomprises at least about 100 proteins.
 26. The method of claim 10wherein using the mass of the signature peptide to identify the proteinfrom which the signature peptide was derived comprises comparing themass of the signature peptide with the masses of reference peptidesderived from putative proteolytic cleavage of a plurality of referenceproteins in a database, wherein at least one reference peptide comprisesat least one affinity ligand.
 27. The method of claim 26 whereinpeptides derived from proteolytic cleavage of the plurality of referenceproteins are, prior to comparing the mass of the signature peptide withthe masses of the reference peptides, computationally selected toexclude reference peptides that do not contain an amino acid upon whichthe affinity selection is based.
 28. The method of claim 6 wherein theprotein is in regulatory flux in response to a stimulus, and wherein thefirst sample is obtained from the biological environment beforeapplication of the stimulus and the second sample is obtained from thebiological environment after application of the stimulus.
 29. The methodof claim 6 wherein the first and second samples are obtained fromdifferent organisms, cells, organs, tissues or bodily fluids, the methodfurther comprising determining differences in concentration of at leastone protein in the organisms, cells, organs, tissues or bodily fluidsfrom which the samples were obtained.
 30. The method of claim 1 furthercomprising identifying a plurality of isotopically labeled proteinshaving substantially the same isotope ratios, wherein the existence ofsaid plurality of isotopically labeled proteins is indicative that theproteins are co-regulated.
 31. The method of claim 6 further comprisingidentifying a plurality of isotopically labeled peptides havingsubstantially the same isotope ratios, wherein the existence of saidplurality of isotopically labeled peptides is indicative that thepeptides are derived from the same protein, or from proteins that areco-regulated.
 32. The method of claim 6 wherein the samples are obtainedfrom a biological environment, and wherein the first sample is obtainedfrom the biological environment before application of a stimulus and thesecond sample is obtained from the biological environment afterapplication of the stimulus.